Compounds and methods for fluorescent labeling

ABSTRACT

Dye reagents useful in labeling biological materials are provided along with methods for their use.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/317,875, filed Sep. 7, 2001 entitled “Compounds and Methods forFluorescent Labeling” (Attorney Docket No. 17682A-007300US), thecontents of which are incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK BACKGROUND OF THE INVENTION

The non-radioactive detection of biological analytes is an importanttechnology in modern analytical biotechnology. By eliminating the needfor radioactive labels, safety is enhanced and the environmental impactof reagent disposal is greatly reduced, resulting in decreased costs foranalysis. Examples of methods utilizing such non-radioactive detectionmethods include DNA sequencing, oligonucleotide probe methods, detectionof polymerase-chain-reaction products, immunoassays, and the like.

In many applications the independent detection of multiple spatiallyoverlapping analytes in a mixture is required, e.g., single-tubemultiplex DNA probe assays, immuno assays, multicolor DNA sequencingmethods, and the like. In the case of multi-loci DNA probe assays, byproviding multicolor detection, the number of reaction tubes may bereduced thereby simplifying the experimental protocols and facilitatingthe manufacturing of application-specific kits. In the case of automatedDNA sequencing, multicolor labeling allows for the analysis of all fourbases in a single lane thereby increasing throughput over single-colormethods and eliminating uncertainties associated with inter-laneelectrophoretic mobility variations.

Multiplex detection imposes a number of severe constraints on theselection of dye labels, particularly for analyses requiring anelectrophoretic separation and treatment with enzymes, e.g., DNAsequencing.

Due to the variety of constraints imposed of the labeling of biologicalmaterials, methodology that is broadly applicable to a variety of dyesis highly desirable. Surprisingly, the present invention provides suchmethodology, along with reagents that are useful in carrying outlabeling processes.

BRIEF SUMMARY OF THE INVENTION

In a broad sense, the present invention provides a method for preparinga fluorescent dye-labeled biological agent, the method comprisingcontacting an unlabeled biological agent with a fluorescent dye-fusedlactone derivative under conditions sufficient to covalently attach thefluorescent dye to said biological agent and form a fluorescentdye-labeled biological agent. In a related aspect, the lactone dyes canbe used to prepare phosphoramidite reagents suitable for labelingbiological agents or materials in, for example, automated synthesizers.

The fluorescent dye-fused lactone derivatives are generally provided ashaving the formula selected from the group consisting of:

wherein R¹ and R^(1′) are each independently selected from H, halogen,cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl andheteroaryl; R⁵, R⁶, R⁷ and R⁸ are each independently selected from H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹, R^(1′),and R⁵ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of R¹, R^(1′),and R⁵ through R⁸ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; R^(A) and R^(B) arecombined to form a substituted or unsubstituted fused ring system havingfrom 1 to 4 five- or six-membered rings; with the proviso that thecompound has an emission wavelength of from 400 nm to 1200 nm.

Preferred fluorescent dye-fused lactone derivatives are based on knowndyes selected from the coumarins, benzocoumarins, xanthenes,benzo[a]xanthenes, benzo[b]xanthenes, benzo[c]xanthenes, phenoxazines,benzo[a]phenoxazines, benzo[b]phenoxazines and benzo[c]phenoxazines.

Additional reagents and methods are provided in which the lactone dyesare converted to phosphoramidite reagents as well as solid support boundlabels useful in fluorescent labeling processes.

The reagents provided herein are stable and in many instances areprovided with certain protecting groups that provide the reagent with along shelf life, making them useful to those engaged in a variety ofresearch efforts.

BRIEF DESCRIPTION OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the meanings given below:

The term “alkyl” refers to a linear, branched, or cyclic saturatedmonovalent hydrocarbon radical or a combination of cyclic and linear orbranched saturated monovalent hydrocarbon radicals having the number ofcarbon atoms indicated in the prefix. For example, (C₁-C₈)alkyl is meantto include methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl,cyclopentyl, cyclopropylmethyl and the like. For each of the definitionsherein (e.g., alkyl, alkenyl, alkoxy, arylalkoxy), when a prefix is notincluded to indicate the number of main chain carbon atoms in an alkylportion, the radical or portion thereof will have eight or fewer mainchain carbon atoms.

The term “alkylene” means a linear saturated divalent hydrocarbonradical or a branched saturated divalent hydrocarbon radical having thenumber of carbon atoms indicated in the prefix. For example,(C₁-C₆)alkylene is meant to include methylene, ethylene, propylene,2-methylpropylene, pentylene, and the like.

The term “alkenyl” refers to a linear monovalent hydrocarbon radical ora branched monovalent hydrocarbon radical having the number of carbonatoms indicated in the prefix and containing at least one double bond.For example, (C₂-C₆)alkenyl is meant to include, ethenyl, propenyl, andthe like.

The term “alkynyl” refers to a linear monovalent hydrocarbon radical ora branched monovalent hydrocarbon radical containing at least one triplebond and having the number of carbon atoms indicated in the prefix. Forexample, (C₂-C₆)alkynyl is meant to include ethynyl, propynyl, and thelike.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively. Similarly, the term dialkylaminorefers to an amino group having two attached alkyl groups that can bethe same or different.

The term “aryl” means a monovalent monocyclic or bicyclic aromatichydrocarbon radical of 6 to 10 ring atoms which is unsubstituted orsubstituted independently with one to four substituents, preferably one,two, or three substituents selected from alkyl, cycloalkyl,cycloalkyl-alkyl, halo, cyano, hydroxy, alkoxy, amino, acylamino,mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, COR(where R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl cut, phenyl orphenylalkyl, aryl or arylalkyl), —(CR′R″)_(n)—COOR (where n is aninteger from 0 to 5, R′ and R″ are independently hydrogen or alkyl, andR is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl cut, phenyl orphenylalkyl aryl or arylalkyl) or —(CR′R″)_(n)—CONR^(a)R^(b) (where n isan integer from 0 to 5, R′ and R″ are independently hydrogen or alkyl,and R^(a) and R^(b) are, independently of each other, hydrogen, alkyl,cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl, aryl or arylalkyl).More specifically the term aryl includes, but is not limited to, phenyl,biphenyl, 1-naphthyl, and 2-naphthyl, and the substituted forms thereof.Similarly, the term “heteroaryl” refers to those aryl groups wherein oneor more heteroatoms or heteroatom functional groups have replaced a ringcarbon, while retaining aromatic properties, e.g., pyridyl, quinolinyl,quinazolinyl, thienyl, and the like. For brevity, the term aryl, whenused in combination with other radicals (e.g., aryloxy, arylalkyl) ismeant to include both aryl groups and heteroaryl groups as describedabove.

The term “arylalkyl” refers to a radical —R^(a)R^(b) where R^(a) is analkylene group (having the indicated number of carbon atoms, or ifunspecified having six or fewer main chain carbon atoms) and R^(b) is anaryl group as defined herein. Examples of arylalkyl groups includebenzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the like.

Similarly the term “arylalkenyl” means a radical —R^(a)R^(b) where R^(a)is an alkenylene group and R^(b) is an aryl group as defined herein,e.g., 3-phenyl-2-propenyl, and the like.

“Arylheteroalkyl” means a radical —R^(a)R^(b) where R^(a) is anheteroalkylene group (having the indicated number of carbon atoms) andR^(b) is an aryl group as defined herein, e.g.,2-hydroxy-2-phenyl-ethyl, 2-hydroxy-1-hydroxymethyl-2-phenyl-ethyl, andthe like.

The term “aryloxy”, refers to a radical —OR where R is an aryl group,e.g., phenoxy, naphthyloxy and the like.

The prefix “halo” and the term “halogen” when used to describe asubstituent, refer to —F, —Cl, —Br and —I.

The term “heteroalkyl” refers to an alkyl radical as defined herein withone, two or three substituents independently selected from cyano,—OR^(a), —NR^(b)R^(c), and —S(O)_(n)R^(d) (where n is an integer from 0to 2), with the understanding that the point of attachment of theheteroalkyl radical is through a carbon atom of the heteroalkyl radical.R^(a) is hydrogen, alkyl, aryl, arylalkyl, alkoxycarbonyl,aryloxycarbonyl, carboxamido, or mono- or di-alkylcarbamoyl. R^(b) ishydrogen, alkyl, aryl or arylalkyl. R^(c) is hydrogen, alkyl, aryl,arylalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- ordi-alkylcarbamoyl or alkylsulfonyl. R^(d) is hydrogen (provided that nis O), alkyl, aryl, arylalkyl, amino, mono-alkylamino, di-alkylamino, orhydroxyalkyl. Representative examples include, for example,2-hydroxyethyl, 2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl,2-cyanoethyl, and 2-methylsulfonyl-ethyl. For each of the above, R^(a),R^(b), R^(c), and R^(d) can be further substituted by NH₂, fluorine,alkylamino, di-alkylamino, OH or alkoxy. Additionally, the prefixindicating the number of carbon atoms (e.g., C₁-C₁₀) refers to the totalnumber of carbon atoms in the portion of the heteroalkyl group exclusiveof the cyano, —OR^(a), —NR^(b)R^(c), or —S(O)_(n)R^(d) portions.

The term “heterocyclic” refers to a saturated or unsaturatednon-aromatic cyclic radical of 3 to 8 ring atoms in which one or tworing atoms are heteroatoms selected from O, NR (where R is independentlyhydrogen or alkyl) or S(O)_(n) (where n is an integer from 0 to 2), theremaining ring atoms being C, where one or two C atoms may optionally bereplaced by a carbonyl group. The heterocyclyl heterocyclic ring may beoptionally substituted independently with one, two, or threesubstituents selected from alkyl, halo, cyano, hydroxy, alkoxy, amino,mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, —COR (where R ishydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl cut, phenyl or phenylalkylaryl or arylalkyl), —(CR′R″)_(n)—COOR (n is an integer from 0 to 5, R′and R″ are independently hydrogen or alkyl, and R is hydrogen, alkyl,cycloalkyl, cycloalkyl-alkyl cut, phenyl or phenylalkyl), or—(CR′R″)_(n)—CONR^(a)R^(b) (where n is an integer from 0 to 5, R′ and R″are independently hydrogen or alkyl, and R^(a) and R^(b) are,independently of each other, hydrogen, alkyl, phenyl or phenylalkyl).More specifically the term heterocyclyl heterocyclic includes, but isnot limited to, tetrahydropyranyl, piperidino, N-methylpiperidin-3-yl,piperazino, N-methylpyrrolidin-3-yl, 3-pyrrolidino, 2-pyrrolidon-1-yl,morpholino, thiomorpholino, thiomorpholino-1-oxide,thiomorpholino-1,1-dioxide, pyrrolidinyl, and the derivatives thereofThe prefix indicating the number of carbon atoms (e.g., C₃-C₁₀) refersto the total number of carbon atoms in the portion of the heterocyclylheterocyclic group exclusive of the number of heteroatoms.

The terms “heteroalkylene” means a linear saturated divalent hydrocarbonradical of one to six carbons or a branched saturated hydrocarbonradical of three to six carbon atoms with one, two or three substituentsindependently selected from —OR^(a), —NR^(b)R^(c), and —S(O)_(n)Rd(where n is an integer from 0 to 2) where, R^(a), R^(b), R^(c), andR^(d) are as defined herein for a heteroalkyl radical. Examples include,2-hydroxyethan-1,2-diyl, 2-hydroxypropan-1,3-diyl and the like.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” and “aryl”) aremeant to include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, heterocycloalkyl, and heterocycloalkenyl) can bea variety of groups selected from: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂ in a numberranging from zero to four, preferably, zero, one, two or threesubstituents. R′, R″ and R′″ each independently refer to hydrogen,unsubstituted (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl, arylsubstituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxygroups, or aryl-(C₁-C₄)alkyl groups. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” in its broadest sense is meant to include groups such ashaloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃,—C(O)CH₂OCH₃, and the like). Preferably, the alkyl groups will have from0-3 substituents, more preferably 0, 1, or 2 substituents, unlessotherwise specified.

Similarly, substituents for the aryl groups are varied and are selectedfrom: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen orunsubstituted (C₁-C₆)alkyl.

Certain compounds or oligonucleotides of the present invention may existin a salt form. Such salts include base addition salts such as sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When the compounds or modified oligonucleotides of thepresent invention contain relatively basic functionalities, acidaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired acid, either neat orin a suitable inert solvent. Examples of acceptable acid addition saltsinclude those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from organic acids like acetic, propionic,isobutyric, maleic, malonic, lactic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention. The methods forthe determination of stereochemistry and the separation of isomers arewell-known in the art (see discussion in Chapter 4 of ADVANCED ORGANICCHEMISTRY, 4th edition J. March, John Wiley and Sons, New York, 1992).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not (e.g,2H), are intended to be encompassed within the scope of the presentinvention.

“Protecting group” or “protected form thereof” refers to a grouping ofatoms that when attached to a reactive group in a molecule masks,reduces or prevents that reactivity. Examples of protecting groups canbe found in T. W. Greene and P. G. Wuts, PROTECTIVE GROUPS IN ORGANICCHEMISTRY, (Wiley, 2nd ed. 1991), Beaucage and Iyer, Tetrahedron 48:2223-2311 (1992), and Harrison and Harrison et al., COMPENDIUM OFSYNTHETIC ORGANIC METHODS, Vols. 1-8 (John Wiley and Sons. 1971-1996).Representative amino protecting groups include formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl(Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES),trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC)and the like (see also, Boyle, A. L. (Editor), CURRENT PROTOCOLS INNUCLEIC ACID CHEMISTRY, John Wiley and Sons, New York, Volume 1, 2000).Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated such as benzyl and trityl ethersas well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers. Additionally, hydroxy groups can be protected byphotoremovable groups such as a-methyl-6-nitopiperonyloxycarbonyl(McGall, G. H. and Fidanza, J. A., Photolithographic synthesis ofhigh-density olignucleotide arrays, in DNA ARRAYS METHODS AND PROTOCOLS,Edited by Rampal J. B., METHODS IN MOLECULAR BIOLOGY, 170: 71-101(2001), Humana Press, Inc., NY; Boyle, Ann L. (Editor), CurrentProtocols in Nucleic Acid Chemistry, John Wiley and Sons, New York,Volume 1, 2000.)

“Optional” or “optionally” in the above definitions means that thesubsequently described event or circumstance may but need not occur, andthat the description includes instances where the event or circumstanceoccurs and instances in which it does not. For example, “heterocyclogroup optionally mono- or di-substituted with an alkyl group” means thatthe alkyl may but need not be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

The term “biological agent” refers to essentially any nucleoside,oligonucleotide, peptide, protein, aminocarbohydrate or ligand, as wellas analogs thereof (e.g., oligonucleotides having modified ornon-natural bases).

The term “conjugate” refers to a molecule formed by the covalentattachment of two or more components such as oligonucleotides,fluorophores, quenchers, minor groove binders, and the like.

“Oligonucleotide” and “polynucleotide” are used interchangeably andrefers to a polymer of nucleotides, either natural or syntheticincluding, but not limited to those nucleotides having modified bases,sugar analogs, and the like. As noted above, an oligonucleotideconjugate will refer to an oligonucleotide as defined, having at leastone covalently attached fluorophore, quencher, minor groove binder (MGB)or other useful fragments, as well as combinations of the recitedcomponents.

The term “solid support” refers to essentially any solid or semisolidmatrix that is useful for, and compatible with, automatedoligonucleotide techniques and includes, glass, polystyrene, nylon,plastic, combinations and the like. Examples of useful solid supportshave been described in, for example, U.S. Pat. Nos. 5,262,530,5,419,966, 5,512,667 and 5,589,586.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques in organic chemistry, biochemistry,oligonucleotide synthesis and modification, bioconjugate chemistry,nucleic acid hybridization, molecular biology, microbiology, genetics,recombinant DNA, and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Maniatis, Fritsch & Sambrook, MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory Press (1982); Sambrook, Fritsch &Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, ColdSpring Harbor Laboratory Press (1989); Ausubel, et al, CURRENT PROTOCOLSIN MOLECULAR BIOLOGY, John Wiley & Sons (1987, 1988, 1989, 1990, 1991,1992, 1993, 1994, 1995, 1996); Gait (ed.), OLIGONUCLEOTIDE SYNTHESIS: APRACTICAL APPROACH, IRL Press (1984); Eckstein (ed.), OLIGONUCLEOTIDESAND ANALOGUES: A PRACTICAL APPROACH, IRL Press (1991).

General

The present invention resides in the discovery that a wide variety offluorescent dyes (or fluorophores) can be prepared having a fusedlactone functional group, providing reagents that are shelf-stable andthat can be used to label essentially any biological agent (e.g.,oligonucleotides, peptides, proteins, probes, and the like) due to theirreactivity with suitable preferably nitrogen-containing (like in nextscheme) nucleophiles. Accordingly, the invention provides new “lactonedyes” as well as methods of labeling biological agents using these“lactone dyes”. The invention further provides reagents such asphosphoramidite-derivatized dyes that can be prepared from the lactonedyes described herein. Additionally, support-bound dyes, similarlyprepared from the lactone dyes are also described.

The “lactone dye” approach to labeling as well as reagent (e.g.,support-bound dyes and phosphoramidites) has been found to be compatiblewith, for example, coumarin dyes, benzocoumarin dyes, fluorescein dyes,rhodol dyes, phenoxazine dyes, benzophenoxazine dyes, xanthene dyes,benzoxanthene dyes, and cyanine dyes.

Examples of these and other suitable dye classes can be found inHaugland, et al., HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS,SIXTH ED., Molecular Probes, Eugene, Ore. 1996; U.S. Pat. Nos.5,187,288; 5,188,934; 5,227,487, 5,248,782; 5,304,645; 5,433,896;5,442,045; 5,556,959; 5,583,236; 5,808,044; 5,986,086; 6,020,481;6,162,931; and 6,221,604; Smith, et al., J. Chem. Soc. Perkin Trans. 2,1993, 1195-1204; Whitaker, et al., Anal. Biochem. 207: 267-279 (1992);and Hirschberg, et al., Biochemistry 37: 10381-10385 (1998).

EMBODIMENTS OF THE INVENTION

Methods for Labeling Biological Agents

In one aspect, the present invention provides methods for preparing afluorescent dye-labeled biological agent, the method comprisingcontacting an unlabeled biological agent with a fluorescent dye-fusedlactone derivative under conditions sufficient to covalently attach thefluorescent dye to said biological agent and form a fluorescentdye-labeled biological agent. The term “fluorescent dye-fused lactonederivative” or more simply “lactone dye” as used herein refers toessentially any fluorescent dye which has a fused 5-, 6-, or 7-memberedlactone (e.g., shares two carbon atoms and one oxygen with the dye)attached to the dye framework. In most instances below, a lactone isillustrated that is a preferred 6-membered lactone, although theinvention is not so limited.

As noted above, the present invention finds broad application inlabeling of nucleic acids (including nucleotides, nucleosides, DNA, RNA,PNA, locked nucleic acids, oligonucleotides and the like), peptides orproteins, oligosaccharides, glycosylated proteins, and other biologicalagents. Additionally, the nucleic acids can include modified bases(e.g., 5-substituted pyrimidines, 3-substituted purines, substituteddeazapurines, substituted pyrazolo[3,4-d]pyrimidines, and the like).See, for example, co-pending application Ser. Nos. 09/724,988 and09/447,936. The invention also finds utility in labeling ofoligonucleotides and modified oligonucleotides having attached groupssuch as minor groove binders, intercalators, crosslinking groups, andthe like.

Lactone Dyes

As noted above, the invention is broadly applicable to the preparationand use of new lactone derivatives of a variety of dyes. In manyembodiments, lactone derivatives can be prepared based on any dye thatcan form a chroman-2-one fragment (fused lactone) as illustrated below.Reaction between a nitrogen-containing substrate or biological agent anda lactone forms a covalent bond on ring opening. Of course, one of skillin the art will appreciate that the lactone methodology (e.g., thesynthesis of a lactone ring fused to a dye) described herein can beapplied to essentially any dye having a ring structure and availablevalence sites for the lactone ring.

Typically, the nucleophiles are nitrogen nucleophiles (e.g., amines,hydrazines and the like) and the ligand (R^(M)) can be a biologicalcompound (e.g., a nucleic acid, peptide, protein and the like) or alinking group that is used to attached the fluorescent molecule to, forexample, a phosphoramidite group or a solid support.

In view of the above, the present invention provides methods forlabeling biological materials and methods for preparing phosphoramiditereagents or solid support reagents by reacting a fluorescent dyefused-lactone derivative having a formula selected from:

with a suitable nucleophile to attach the fluorescent dye to a linkinggroup, solid support, or biological material. In the above formulae, R¹and R^(1′) are each members independently selected from the groupconsisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio,(C₁-C₈)alkoxy, aryl and heteroaryl; R⁵, R⁶, R⁷ and R⁸ are eachindependently selected from the group consisting of H, (C₁-C₈)alkyl,aryl, heteroaryl, aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; whereinthe alkyl portions of any of R¹, R^(1′), or R⁵ through R⁸ are optionallysubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms; and the aryl or heteroarylportions of any of R¹, R^(1′), and R⁵ through R⁸ are optionallysubstituted with from one to four substituents selected from the groupconsisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy.Additionally, the symbols R^(A) and R^(B) are combined to form asubstituted or unsubstituted fused ring system having from 1 to 4 five-or six-membered rings; with the proviso that the compound has anemission wavelength of from 400 nm to 1200 nm, more preferably, 400 nmto about 850 nm.

While the present invention finds broad application to a number ofchroman-2-one dyes, certain groups of dyes are preferred and areoutlined below.

A. Xanthenes (Fluoresceins and Rhodols)

The methods of the present invention can be carried out using xanthenefused-lactone dyes having the formula:

wherein A¹ represents O or N—Z in which Z is H or (C₁-C₈)alkyl, or isoptionally combined with R² or R⁴ to form a 5- or 6-membered ring or iscombined with each of R² and R⁴ to form two fused 6-membered rings; R¹,R^(1′), R², R³ and R⁴ are each independently selected from H, halogen,cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl andheteroaryl; R⁵, R⁶, K7 and R⁸ are each independently selected from thegroup consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyland heteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′)or R¹ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of R^(1′) andR¹ through R⁸ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; R⁰ is halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl or heteroarylhaving the formula:

-   -   wherein X¹, X², X³, X⁴ and X⁵ are each independently selected        from the group consisting of H, halogen, cyano, CF₃,        (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl,        (C₁-C₈)alkynyl, SO₃H and CO₂H. Additionally, the alkyl portions        of any of X¹ through X⁵ can be further substituted with halogen,        carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano,        haloacetyl or hydroxy, and the alkyl portions of the        substituents have from 1 to 6 carbon atoms. Optionally, any two        adjacent substituents X¹ through X⁵ can be taken together to        form a fused aromatic ring that is optionally further        substituted with from one to four substituents selected from        halogen cyano, carboxy, sulfo, hydroxy, amino, mono- or        di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and        (C₁-C₆)alkoxy.

In certain embodiments, the lactone dyes of formula I (as well as otherformulae herein) will be present in isomeric or tautomeric forms (e.g.,spirolactones that result from compounds in which R⁰ is substitutedphenyl or pyridyl and X⁵ is CO₂H or SO₃H) and are included in thisinvention.

When the tautomeric form has a hydroxy (or amino) group positionedbetween R² and R⁴, protected forms are also contemplated by the presentinvention. Thus, for those formulae in the present invention wherein A¹is provided as O and NR, the invention further includes those compoundsin which A is OH or a protected hydroxy group, and NH₂ or a protectedamino group.

Accordingly, in one group of preferred embodiments, the xanthenefused-lactone dyes are fluorescein fused-lactone dyes selected fromthose having one of the following formulae:

wherein A represents a hydroxy, amino, protected hydroxy, or protectedamino, or is optionally combined with R² or R⁴ to form a 5- or6-membered ring or is combined with each of R² and R⁴ to form two fused6-membered rings; R^(1′), R¹, R², R³ and R⁴ are each independentlyselected from H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and(C₁-C₈)alkoxy; R⁵, R⁶, R⁷ and R⁸ are each independently selected from H,(C₁-C₈)alkyl, aryl and aryl(C₁-C₄)alkyl; wherein the alkyl portions ofany of R¹ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl portions of any of R⁵ through R⁸ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy. Further preferred are those compounds of formulae Ia andIb wherein A is hydroxy or a protected hydroxy. Preferred protectinggroups are acyl groups derived from (C₂-C₂₀)alkanoic acids (e.g.,acetyl, propionyl, pivaloyl, isobutyryl, and the like). Still furtherpreferred are those compounds having a formula above in which R^(1′) andR¹ through R⁸ are independently selected from H, halogen, CF₃ and cyano.

The fused-lactone fluorescein dyes of the present invention cangenerally be prepared according to the scheme below, in which a suitablysubstituted resorcinol (i) is reacted with a substituted benzophenone(ii), then lactonized to produce the desired compounds (iii). Certainsubstituents are not included in the formula below. A more detailedreaction scheme and a table of particularly preferred lactone dyes inthis group are provided in the section entitled “General Synthesis ofLactone Dyes.”

B. Benzo[a]xanthenes

Benzo[a]xanthene fused lactone dyes that are useful in the presentmethods are provided having the formula:

wherein A¹ represents O or N—Z in which Z is H or (C₁-C₈)alkyl, or isoptionally combined with R⁴ to form a 5- or 6-membered ring; R¹, R^(1′)and R⁴ are each independently selected from H, halogen, cyano, CF₃.(C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; thesubscript n is 0 to 4 and each X⁰ is independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl, heteroaryl, SO₃Hand CO₂H; R⁵, R⁶, R⁷ and R⁸ are each independently selected from thegroup consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyland heteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of X⁰,R^(1′) or R¹ through R⁸ are optionally substituted with halogen,carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetylor hydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of X⁰, R^(1′)and R¹ through R⁸ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; R° is halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl or heteroarylhaving the formula:

In formula II, as well as the aryl and heteroaryl substituents above,each X¹, X², X³, X⁴ and X⁵ is independently selected from the groupconsisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, SO₃H and CO₂H.Additionally, the alkyl portions of any of X¹ through X⁵ can be furthersubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms. Optionally, any two adjacentsubstituents X¹ through X⁵, or two adjacent X⁰ groups can be takentogether to form a fused aromatic ring that is optionally furthersubstituted with from one to four substituents selected from halogencyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy. The subscript n is aninteger of from 0 to 4.

In a preferred group of embodiments, the fused-lactone benzo[a]xanthenedyes are those having a formula:

wherein A is hydroxy or protected hydroxy, or amino or protected amino;each X⁰ and n has the meaning given with reference to formula II, andeach of X¹, X², X³, X⁴, R¹, R^(1′), R⁴, and R⁵ through R⁸ have themeanings provided above with reference to formulae I. Further preferredare those compounds of formulae Ia and IIc wherein A is hydroxy or aprotected hydroxy. Preferred protecting groups are acyl groups derivedfrom (C₂-C₂₀)alkanoic acids (e.g., acetyl, propionyl, pivaloyl,isobutyryl, and the like). More preferably, R^(1′) and R¹ through R⁸ areindependently selected from H, halogen, CF₃ and cyano; and each X⁰ is H,halogen, CF₃ or cyano. In other preferred embodiments, two of X¹ throughX⁴ are combined to form a six-membered aromatic ring. In otherparticularly preferred embodiments, in the compounds of IIa and IIc,each of R⁵ through R⁸ is H. Most preferred are those embodiments inwhich A is hydroxy or protected hydroxy; each of R⁵ through R⁸ is H;each of X¹ through X⁴ is H, F or Cl; and R¹, R^(1′), and R⁴ are eachindependently selected from H, F, Cl, CN and CF₃.

In general, these compounds can be prepared according to the procedurein Reaction Scheme 2.

C. Benzo[b]xanthenes

Still other compounds useful in the present invention are thefused-lactone benzo[b]xanthenes provided in formulae (III) and (III′)below.

In each of these formulae, A¹, R⁰ through R⁸, X⁰ are as described abovewith reference to formula II and the subscript n is an integer of from 0to 3. Optionally A¹ can be combined with an adjacent X⁰ group to form a5- or 6-membered ring or can be combined with two adjacent X⁰ groups toform two fused 6-membered rings.

Particularly preferred fused-lactone benzo[b]xanthenes are provided informula (IIIa) and (IIIb):

wherein A represents a hydroxy, amino, protected hydroxy, protectedamino; R^(1′), R¹, R³ and R⁴ are each independently selected from H,halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy;R⁵, R⁶, R⁷ and R⁸ are each independently selected from H, (C₁-C₈)alkyl,aryl and aryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′),and R¹ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl portions of any of R⁵ through R⁸ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy. The remaining substituents (X⁰, X¹ through X⁴) have themeanings provided with reference to general formulae III and III′.

In further preferred embodiments, the present methods employ thosecompounds of formulae IIIa or IIIb wherein A is hydroxy or a protectedhydroxy. Preferred protecting groups are acyl groups derived from(C₂-C₂₀)alkanoic acids (e.g., acetyl, propionyl, pivaloyl, isobutyryl,and the like). Still further preferred are those compounds of formulaeIIIa or IIIb in which R^(1′) and R¹ through R⁸ are independentlyselected from H, halogen, CF₃ and cyano. More preferably, each X⁰ is H,halogen, CF₃ or cyano. In other preferred embodiments, two of X¹ throughX⁴ are combined to form a six-membered aromatic or heteroaromatic ring.In other particularly preferred embodiments, in the compounds of IIIaand IIIb, each of R⁵ through R⁸ is H.

D. Benzo[c]xanthenes

Still other fused-lactone benzo[c]xanthenes that are useful in thepresent invention are provided in formulae (IV) and (IV′):

wherein A¹, R⁰ through R⁸ are as described with reference to formula Iand X⁰ and n are as described above with reference to formulae II andIII. Optionally A¹ can be combined with an adjacent X⁰ group to form a5- or 6-membered ring or can be combined with two adjacent X⁰ groups toform two fused 6-membered rings.

Particularly preferred fused-lactone benzo[b]xanthenes are provided informulae (IVa) through (IVd):

wherein A represents a hydroxy, amino, protected hydroxy, protectedamino; R^(1′), R¹, R², R³ and R⁴ are each independently selected from H,halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy;R⁵, R⁶, R⁷ and R⁸ are each independently selected from H, (C₁-C₈)alkyl,aryl and aryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′),and R¹ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl portions of any of R⁵ through R⁸ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy. The remaining substituents (X⁰, X¹ through X⁴) have themeanings provided with reference to general formula IV and IV′.

In further preferred embodiments, the present methods employ thosecompounds of formulae IVa or IVb wherein A is hydroxy or a protectedhydroxy. Preferred protecting groups are acyl groups derived from(C₂-C₂₀)alkanoic acids (e.g., acetyl, propionyl, pivaloyl, isobutyryl,and the like). Still further preferred are those compounds of formulaeIVa or IVb in which R^(1′) and R¹ through R⁸ are independently selectedfrom H, halogen, CF₃ and cyano. More preferably, each X⁰ is H, halogen,CF₃ or cyano. In other preferred embodiments, two of X¹ through X⁴ arecombined to form a six-membered aromatic or heteroaromatic ring. Inother particularly preferred embodiments, in the compounds of IVa andIVb, each of R⁵ through R⁸ is H.

E. Phenoxazines

In still another group of embodiments, the fused-lactone dyes arerepresented by the formulae (V) and (V′):

in which the symbols A¹, and R¹ through R⁸ have the meanings providedabove with respect to compounds of formula II, III and IV. Optionally A¹can be combined with R² or R⁴ to form a 5- or 6-membered ring or can becombined with each of R² and R⁴ to form two fused 6-membered rings.Additionally, R⁹ can be any of the groups provided for R^(1′).

In certain preferred embodiments, R¹, R², R³, R⁴ and R⁹ are eachindependently selected from H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio and (C₁-C₈)alkoxy; R⁵, R⁶, R⁷ and R⁸ are eachindependently selected from H, (C₁-C₈)alkyl, aryl and aryl(C₁-C₄)alkyl;wherein the alkyl portions of any of R¹ through R⁹ are optionallysubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms; and the aryl portions of anyof R⁵ through R⁸ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy. Still furtherpreferred are those compounds of formula V in which R¹ through R⁹ areindependently selected from H, halogen, CF₃ and cyano. In otherparticularly preferred embodiments, in the compounds of V and V′, eachof R⁵ through R⁸ is H and each of R¹ through R⁴ and R⁹ is independentlyselected from the group consisting of hydrogen and halogen, morepreferably, H, Cl and F.

The fused-lactone phenoxazine dyes of the present invention cangenerally be prepared according to the scheme below, in which a suitablysubstituted nitrosoresorcinol (vii) is reacted with a suitablysubstituted dihydroxy hydrocinnamic acid (viii), then lactonized toproduce the desired compounds (ix). Certain substituents are notincluded in the formula below. A more detailed reaction scheme and atable of particularly preferred lactone dyes in this group are providedin the section entitled “General Synthesis of Lactone Dyes.”

F. Benzo[a]phenoxazines

In a group of related embodiments, the fused lactone dyes are based onthe benzo[a]phenoxazine dyes and are represented by the formulae (VI)and (VI′):

wherein A¹ represents O or N—Z in which Z is H or (C₁-C₈)alkyl, or isoptionally combined with R⁴ to form a 5- or 6-membered ring; R¹, R⁴ andR⁹ are each independently selected from H, halogen, cyano, CF₃,(C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; R⁵,R⁶, R⁷ and R⁸ are each independently selected from the group consistingof H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹ or R⁴through R⁹ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl or heteroaryl portions of any of R¹ and R⁴ through R⁹ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; each X⁰ is independently selected from the groupconsisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₆)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl, heteroaryl, SO₃Hand CO₂H. Additionally, the alkyl or aryl portions of any X⁰ can befurther substituted with halogen, carboxy, sulfo, amino, mono- ordialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms. Optionally,any two adjacent X⁰ groups can be taken together to form a fusedaromatic or heteroaromatic ring that is optionally further substitutedwith from one to four substituents selected from halogen cyano, carboxy,sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl,(C₁-C₆)alkylthio and (C₁-C₆)alkoxy. The subscript n is an integer offrom 0 to 4.

G. Benzo[b]phenoxazines

Still other compounds useful in the present methods are provided informulae VII and VII′:

wherein A¹ represents O or N—Z in which Z is H or (C₁-C₈)alkyl, or iscombined with an adjacent X⁰ group to form a 5- or 6-membered ring orcan be combined with two adjacent X⁰ groups to form two fused 6-memberedrings; R¹, R³, R⁴ and R⁹ are each independently selected from H,halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryland heteroaryl; R⁵, R⁶, K7 and R⁸ are each independently selected fromthe group consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl,aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; wherein the alkyl portionsof any of R¹ or R³ through R⁹ are optionally substituted with halogen,carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetylor hydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of R¹ and R³through R⁹ are optionally substituted with from one to four substituentsselected from the group consisting of halogen, cyano, carboxy, sulfo,hydroxy, amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl,(C₁-C₆)alkylthio and (C₁-C₆)alkoxy; each X⁰ is independently selectedfrom the group consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl,heteroaryl, SO₃H and CO₂H. Additionally, the alkyl or aryl portions ofany X⁰ can be further substituted with halogen, carboxy, sulfo, amino,mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and thealkyl portions of the substituents have from 1 to 6 carbon atoms.Optionally, any two adjacent X⁰ groups can be taken together to form afused aromatic or heteroaromatic ring that is optionally furthersubstituted with from one to four substituents selected from halogencyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy. The subscript n is aninteger of from 0 to 3.

H. Benzo[c]phenoxazines

Still other compounds useful in the present methods are provided informulae VIII and VIII′:

wherein A¹ represents O or N—Z in which Z is H or (C₁-C₈)alkyl, or iscombined with an adjacent X⁰ group to form a 5- or 6-membered ring orcan be combined with two adjacent X⁰ groups to form two fused 6-memberedrings; R¹, R², R³ and R⁹ are each independently selected from H,halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryland heteroaryl; R⁵, R⁶, R⁷ and R⁸ are each independently selected fromthe group consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl,aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; wherein the alkyl portionsof any of R¹, R², R³ or R⁵ through R⁹ are optionally substituted withhalogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano,haloacetyl or hydroxy, and the alkyl portions of the substituents havefrom 1 to 6 carbon atoms; and the aryl or heteroaryl portions of any ofR¹, R², R³ or R⁹ through R⁹ are optionally substituted with from one tofour substituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; each X⁰ isindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl,(C₁-C₈)alkynyl, aryl, heteroaryl, SO₃H and CO₂H. Additionally, the alkylor aryl portions of any X⁰ can be further substituted with halogen,carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetylor hydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms. Optionally, any two adjacent X⁰ groups can be takentogether to form a fused aromatic or heteroaromatic ring that isoptionally further substituted with from one to four substituentsselected from halogen cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy.The subscript n is an integer of from 0 to 3.

I. Coumarins and Benzocoumarins

The methods of the present invention can be carried out using coumarinor benzocoumarin fused-lactone dyes.

Coumarin fused-lactone dyes useful in the invention, have the formula:

wherein R¹, R^(1′), R² and R³ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; R⁵, R⁶, R⁷ and R⁸are each independently selected from the group consisting of H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′) orR¹ through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl or heteroaryl portions of any of R^(1′) and R¹ through R⁸are optionally substituted with from one to four substituents selectedfrom the group consisting of halogen, cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy. In certain embodiments, R² and R³ are taken together toform a fused aromatic ring. The symbol A¹ represents O or N—Z, in whichZ is H or (C₁-C₈)alkyl.

In certain embodiments, R² and R³ are independently selected fromhalogen, cyano, CF₃, (C₁-C₈)alkyl, and aryl or heteroaryl having theformula:

wherein X¹, X², X³, X⁴ and X⁵ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, SO₃H and CO₂H.Additionally, the alkyl portions of any of X¹ through X⁵ can be furthersubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms. Optionally, any two adjacentsubstituents X¹ through X⁵ can be taken together to form a fusedaromatic ring that is optionally further substituted with from one tofour substituents selected from halogen cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy.

The benzocoumarin series of dyes are those of formula IX in which R² andR³ are combined to form a fused benzene ring, optionally substitutedwith one to four substituents selected from halogen cyano, carboxy,sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl,(C₁-C₆)alkylthio and (C₁-C₆)alkoxy.

Certain coumarin fused-lactone dyes can be prepared as outlined inReaction Scheme 4.

General Synthesis of Lactone Dyes

Many of the fused-lactone dyes can be prepared from common intermediatethat are readily available to one of skill in the art. Two groups ofparticularly useful intermediates are the 2-substituted1,3-dihydroxyphenylpropionates and the isomeric, 4-substituted2,6-dihydroxyphenylpropionates. These compounds can be prepared asoutlined below.

2-Substituted 1,3-dihydroxyphenylpropionates 5a-c were synthesized in 4steps (Reaction Scheme 5A) in good total yields starting from either2-chloro-1,3-dimethoxybenzene (1a) (Kovacic, P.; Kurz, M. E. J. Org.Chem. 1966, 31, 2459-2467; Wada, M.; Wakamori, H.; Hirawa, A.; Erabi, T.Bull. Chem. Soc. Jpn., 1992, 65 (5), 1389-1391) or2-fluoro-1,3-dimethoxybenzene (1b) (W.-C. Sun et al. J. Org. Chem. 1997,62, 6469-6475) or 2-phenyl-1,3-dimethoxybenzene (1c) (U.S. Pat. No.6,221,604). Formylation with α,α-dichloromethyl methyl ether in thepresence of TiCl₄ afforded aldehydes 2 in high yields (95%). Thesecompounds were quantitatively converted into the cinnamic acids 3 byreaction with malonic acid in the presence of catalytic amount ofpiperidine. Catalytic hydrogenation (10% Pd/C, 40 psi) gave acids 4 ingreater than 95% yields. Deprotection of the methoxy groups using aceticacid/aqueous hydrobromic acid mixture followed by esterification of thecarboxy group (methanol/HCl gas) afforded the desired 2-substitutedmethyl 1,3-dihydroxyphenylpropionates 5. Compound 5d was prepared by analternative synthetic route in accordance with Blickenstaff, R. T., etal. (Tetrahedron, 24: 2495-2498 (1968)).

The isomeric methyl 2,6-dihydroxyphenylpropionates 30 and 31 weresynthesized starting from 3-(2,6-dihydroxyphenyl)propionic acid (MitoshiK. et al. Syn. Lett., 12: 1472-1474 (1997)) as shown above.

The dihydroxyphenylpropionates have been implemented into the synthesisof several classes of chromanone dye precursors. The syntheticapproaches herein are based on utilizing the versatile substitutedmethyl dihydroxyphenylpropionates in conjunction with known syntheticroutes for dye assembly. The application of the intermediates is notlimited to the classes described below, but finds broad application toany resorcinol based dye chemistry. Moreover, the intermediatesdescribed above can be prepared using a variety of other art-recognizedmethods.

Coumarin Lactone Dye Synthesis

Resorcinol analogs 5 were reacted with diketene to afford8-halocoumarins 6 in high yields. Hydrolysis of the ester groupgenerated free acids 7 which were lactonized in the presence ofpentafluorophenyl trifluoroacetate (PFP-TFA) to give chromanones 8. SeeReaction Scheme 6.Reaction Scheme 6

Phenoxazine Lactone Dye Synthesis

Phenoxazine lactone dyes can be prepared as generally outlined inReaction Scheme 3 and more specifically provided in Reaction Scheme 7,below.

Reaction of 4-nitrosorecorcinol derivatives 9 (commercially available orsynthesized in accordance with the state-of-the-art) and 5 in thepresence of MnO₂ and using methanol as a solvent gave crystallineprecipitates of sufficiently pure N-oxides 10 in satisfactory yields(20-40%). The ester functions were hydrolyzed with NaOH followed byreduction of the N-oxide groups by a treatment with sodium diothioniteto yield resorufin derivatives 11. Treatment of acids 11 with PFP/TFAcatalyzed the formation of lactones 12.

Xanthene Dye Lactone Synthesis

Synthesis of asymmetrical xanthene dyes (Reaction Scheme 8a) wasaccomplished in two stages. At the first stage benzophenones 14 wereprepared in good to excellent yields by Friedel-Crafts acylation ofresorcinol analogs 5 with phthalic anhydrides 13 in the presence ofAlCl₃. At the second stage ketones 14 were reacted with resorcinols 15using either methanesulfonic or trifluoroacetic (plus catalyticmethanesulfonic acid) acid as a solvent. Depending on the solvent eitheresters 16 or free acids 17 were obtained. The acid route was preferredfor consequent conversion into lactones 18, the latest was achieved bytreatment of acids 17 with PFP-TFA.

An alternative synthesis is provided in Scheme 8b for asymmetric4,7-unsubstituted xanthenes (compounds of Scheme 8a, wherein X¹ and X⁴are H). While the two-step condensation method is illustrated for the4,7-unsubstituted class of xanthene dyes, one of skill in the art willappreciate that the method is not so limited and can be applied to moreefficient preparation of many xanthene dyes.

The synthesis of asymmetric 4,7-unsubstituted (X¹ and X⁴=H Scheme 8a)using Scheme 8a provides compounds such as 17c (X¹-X⁴=H) in about 20%yield and requires laborious chromatography purification. The related5,6-dichlorosubstituted fluoresceins synthesized by the same method(Scheme 8a) gave only 20-60% of the desired product. Still otherconventional synthetic procedures such as fusion with ZnCl₂, sulfuricacid or methanesulfonic acid produced similar unsatisfactory results.By-products of the reaction were determined to be symmetric fluoresceinsof structures 33 and 34 (Scheme 8b). Without intending to be bound bytheory, these compounds appear to be formed due to the reversibility ofthe triphenylmethane intermediate at elevated temperatures. It should benoted that that same side products but in smaller amounts (5-10%) werealso observed during the preparation of 4,7-chloro- or fluorosubstitutedfluoresceines (17a-17f).

An alternative, and preferred, synthetic procedure was used for thesynthesis of 4,7-unsubstituted asymmetric xanthene dyes (Scheme 8b). Thereaction between benzophenone 14 and resorcinol 15 was first carried outat low temperatures (0-20° C.) in the presence of methanesulfonic acidas a catalyst and trifluoroacetic acid as a solvent. Under theseconditions only the desired triphenylmethane intermediate is formedwhich can be isolated as a colorless spiro-lactone 32 (Scheme 8b). Thefollowing cyclization step was performed under neutral or mild basicconditions in aqueous solutions at 20-60° C. Finally, the acids 17 wereprecipitated by acidification. This approach allowed the preparation of4,7-unsubstituted asymmetric fluoresceins in excellent yields.

The new method provides a universal way of synthesizing asymmetricxanthene dyes under very mild conditions (e.g., at temperatures of fromabout 0-20° C.) and therefore, is useful for the introduction of morelabile functional groups. The method is particularly useful for thepreparation of asymmetric 4,7-unsubstituted xanthenes which arepractically inaccessible by conventional approaches.

Compounds 16 and 17 synthesized according to the process shown inReaction Scheme 8a and 8b are listed in Table 1 with absorbance maximum(Abs), emission maximum (Em) and Stokes shift (St) in nm. TABLE 1 X¹ andX² and Abs Em No R¹ R² R³ X⁴ X³ (nm) (nm) St (nm) 16c F Cl H Cl Cl 530546 16 16b F Cl H F F 528 546 18 16a Cl Cl H Cl Cl 527 544 17 16d Cl ClH F F 527 543 16 16e H Cl H F F 518 534 16 16f H H H F F 512 530 18 17dCl Cl CH₃ Cl H 531 549 18 17e Cl Cl H Cl H 524 540 16 17f Cl H H Cl H519 534 15 17g Cl Cl H F H 522 539 17 17h Cl Cl CH₃ H Cl 520 543 23 17iCl Cl H H Cl 514 535 21 17j Cl H H H Cl 508 530 22Fluorescence and absorbance spectra were recorded in 0.1 M phosphatebuffer pH 7.5

Reaction Scheme 8c illustrates the preparation of an isomeric xanthenedye (a lactone dye of formula Ib, above.

Benzo[a]- and Benzo[c]xanthene Dye Lactone Synthesis

Benzo[a]- and benzo[c]xanthene dyes 19 (Reaction Scheme 9) and 23(Reaction Scheme 10) were synthesized analogously to xanthene dyes(Reaction Scheme 8) using condensation of benzophenones 14 either with1,3- or 1,6-dihydroxynaphthalene. Two of the esters 19 were hydrolyzedto acids 20 and then converted into lactones 21. Spectral properties ofesters 19 summarized in Table 2.

Particular compounds prepared according to this Scheme are provided inTable 2 along with absorption, emission and Stokes shift wavelengths.TABLE 2 Physical Characteristics of Compounds Synthesized by ReactionScheme 9 X¹ and X² and Abs Em St No R¹ R⁴ X⁴ X³ (nm) (nm) (nm) 19b F H FF 535 570 35 19c F H Cl Cl 537 571 34 19d Cl H F F 535 567 32 19a Cl HCl Cl 538 565 27 19f F H H Cl 522 562 40 19g H H F F 529 560 31 19e Cl HH Cl 522 557 35 19h Cl H H H 516 546 30 19i F H H H 517 551 34

Derivatization of Amino-Tailed Oligonucleotides Using Lactone Dyes.

In order to evaluate the utility of chromanone dye precursors as aminederivatizing agents post-synthetic modification of aminohexyl-tailedoligonucleotides (ODNs) was tested. Four classes of the chromanones,which varied in both chromaphore structure and acidity of leavingphenolic hydroxy group, were used. The reactions between the reagentsand triethylammonium salts of aminohexyl-modified ODNs were done inorganic (DMSO) solvent. Small (2-5×) molar excesses of the reactive dyesled to essentially quantitative (90+%) labeling of the amine-containingligand. The conjugates were purified by gel-electrophoresis andreverse-phase HPLC. Analysis of absorbance and fluorescence spectra(Table 3) of the conjugates demonstrated successful dye incorporationinto the amine-containing ligand and expected spectral characteristics.

TABLE 3 Absorbance and fluorescence properties of dye-modifiedoctathymidylates. Absorbance Extinction Emission Signal Stokes max (nm)coefficient* max (nm) Strength** shift (nm)  8a-T₈ 375 20000 451 0.13 76 8b-T₈ 371 20000 461 0.12 90 18c-T₈ 495 85000 518 1.0 23 18a-T₈ 532100000 548 0.6 16 18b-T₈ 530 100000 546 0.6 16 21b-T₈ 520 65000 552 0.332 21a-T₈ 542 65000 570 0.25 28 12a-T₈ 581 70000 594 0.125 13 12b-T₈ 59170000 604 0.06 13 12c-T₈ 592 70000 603 0.06 11 12d-T₈ 601 65000 616 0.0315 12e-T₈ 601 60000 616 0.015 15*The extinction coefficients were estimated by subtracting absorbancespectra of free dyes from the spectra of corresponding dye-labeledoctathymidylates. The estimate assumes that absorbance at 260 nm is asum of T₈ (molar extinction coefficient at 260 nm = 65000) and dyeabsorbances.**Signal strength was determined as a relative molar fluorescencemeasured at the emission maximum (slit 2.5 nm) with excitation at thecorresponding absorbance maximum (slit 2.5 nm). Fluorescence spectrawere recorded on LS-50B (Perkin-Elmer) luminescence spectrophotometer in0.15 M borate buffer pH 8.5.

In view of the broadly applicable synthetic methodology provided above,the present invention further provides a fused-lactone dye having theformula:

In the above formulae, R¹ and R^(1″) are each members independentlyselected from the group consisting of H, halogen, cyano, CF₃,(C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; R⁵,R⁶, R⁷ and R⁸ are each independently selected from the group consistingof H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹, R^(1′),or R⁵ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of R¹, R^(1′),and R⁵ through R⁸ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy. Additionally, thesymbols R^(A) and R^(B) are combined to form a substituted orunsubstituted fused ring system having from 1 to 4 five- or six-memberedrings; with the proviso that the compound has an emission wavelength offrom 400 nm to 1200 nm, more preferably, 400 nm to about 850 mm.

Preferred groups of fused-lactone dyes are those provided in any offormula I, II, III, IV, V, VI, VII, VIII, IX or X, as well as thepreferred fused-lactone dyes of formula Ia, Ib, IIa, IIb, IIIa, IIIb,IVa and IVb. Particularly preferred fused-lactone dyes are the preferreddyes listing in each of these formulae. Still further preferred arethose fused-lactone dyes prepared and described in detail herein in anyof Reaction Schemes 1 through 10.

Dye Reagents

The fused-lactone dyes provided above can be used to directly labelbiological materials or can be used to prepare a number of dye reagentsthat are useful in the methods described herein, as well as otherlabeling processes.

The scheme below illustrates both the opening of a “dye lactone”(abbreviated as a benzene fused lactone) with an amino ligand, and witha prolinol based linking group that is further elaborated to preparephosphoramidite reagents or solid support bound reagents.

In view of the above, the present invention further provides a methodfor preparing a fluorescent dye-labeled phosphoramidite reagent, saidmethod comprising:

-   -   (a) contacting a fluorescent dye-fused lactone derivative with a        linking group component to form an intermediate fluorescent        dye-labeled linking group; and    -   (b) contacting said intermediate fluorescent dye-labeled linking        group with a phosphoramidite moiety under conditions sufficient        to covalently attach the phosphoramidite moiety to said        fluorescent dye-labeled linking group and form said fluorescent        dye-labeled phosphoramidite reagent. In preferred embodiments,        the fluorescent dye-fused lactone derivative has a formula        selected from the group consisting of I, II, III, IV, V, VI,        VII, VIII, and IX. More preferably, the fluorescent dye-fused        lactone derivative has a formula selected from the group        consisting of Ia, Ib, Ia, IIb, IIIa, IIIb, IVa, IVb, V and VI.        In other preferred embodiments, the linking group component        comprises two reactive functional groups selected from amino,        hydroxy, hydrazino and thiol, and is either linear or cyclic, or        a combination of linear and cyclic. Still further preferred are        those embodiments in which the linking group comprises a        (C₂-C₂₀)alkylene or (C₂-C₂₀)heteroalkylene group. In related and        preferred embodiments, the linking group is cyclic and comprises        a five-membered heterocycle, more preferably the linking group        is a prolinol linker.

In view of the facility with which these phosphoramidite reagents can beprepared, and the further application of this type of methodology topreparing solid support bound dye reagents, the present inventionprovides, in another aspect, dye reagents represented by a formulaselected from:

In the above formulae, R¹ and R^(1′) are each members independentlyselected from the group consisting of H, halogen, cyano, CF₃,(C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; eachR⁵ and R⁶ is independently selected from the group consisting of H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹, R^(1′),R⁵ and R⁶ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl or heteroaryl portions of any of R¹, R^(1′), R⁵ and R⁶ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy. Additionally, the symbols R^(A) and R^(B) are combined toform a substituted or unsubstituted fused ring system having from 1 to 4five- or six-membered rings, optionally substituted with from 1 to 6substituents selected from halogen, cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; R^(x) is selected from the group consisting of H andhydroxy protecting groups; the subscript p is an integer of from 1 to 3;W, or Wand K taken together is a linking group which can be abifunctional linker, trifunctional linker or polyfunctional linker (see,for example, di- and tri-functional linkers described in detail in theliterature, as well as a 3′-alkylamine linker in U.S. Pat. No. 5,419,966and a prolinol-based linker, described in U.S. Pat. No. 5,512,667; tri-and tetrafunctional linkers have also been described in U.S. Pat. Nos.5,451,463, 5,942,610 and 5,696,251; photocleavable linking groups foruse in solid phase synthesis have been described in U.S. Pat. No.5,739,386; trifunctional linkers are also available from Glen Research,Sterling, Va.). Additionally, the linking group W can be acyclic,cyclic, aromatic or a combination thereof, having from 4 to 50 atomsselected from the group consisting of C, N, O, P and S and exclusive ofhydrogen atoms that fill available valences, and further having anitrogen atom directly connected to the adjacent carbonyl group; K isselected from the group consisting of H, OH, SH, NH, (C₁-C₈)alkyl, aryland a nitrogen or hydroxy protecting group, or is combined with W asnoted above (such that in some embodiments, K represents a lone pair ofelectrons); the subscript n is 0 or 1; and when n is 1, Y is a cleavablelinking group and L is a solid support; and when n is 0, L is aphosphoramidite or reactive functional group. The dye reagent willpreferably have an emission wavelength of from 400 nm to 1200 nm, morepreferably, 400 nm to about 850 nm.

In one group of preferred embodiments, n is 1 and Y is a cleavablelinking group selected from the group consisting of:

wherein the subscripts q and r are independently integers of from 1 to15; and each R is independently (C₁-C₈)alkyl or (C₁-C₈)alkoxy.

In another group of preferred embodiments, R^(A), R^(B) and the ring towhich each is attached forms a dye selected from the group consisting ofa coumarin, a benzocoumarin, a xanthene, a benzo[a]xanthene, abenzo[b]xanthene, a benzo[c]xanthene, a phenoxazine, abenzo[a]phenoxazine, a benzo[b]phenoxazine and a benzo[c]phenoxazine.

In one particularly preferred group of embodiments, the dye reagent hasthe formula:

wherein A^(l) is O or N—Z in which Z is H or (C₁-C₈)alkyl; A is O or NH;R^(x) is a protecting group; R¹, R², R³, R⁴ and R⁹ are eachindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl;each R⁵ and R⁶ is independently selected from the group consisting of H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R⁵ throughR⁶ and R⁹ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl portions of any of R¹ through R⁶ and R⁹ are optionallysubstituted with from one to four substituents selected from the groupconsisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;optionally, R² taken together with R³ form a fused aromatic orheteroaromatic ring that is optionally substituted with from one to foursubstituents selected from halogen cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; and tautomeric forms thereof. The letters W, K, Y and L,as well as the subscripts p and n have the meaning provided above withreference to the dye reagents in their general interpretation.

Within this group of embodiments noted above, the dye reagent will morepreferably have the formula:

Reaction Scheme 12 illustrates the preparation of phosphoramiditereagents using chromanone dye precursors.

Resorufin phosphoramidite 28 was prepared from 12a in 2 steps in 50%total yield. At the first step, DMT-protected hydroxyprolinol(synthesized from Fmoc-DMT-hydroxyprolinol) was first efficientlyreacted with lactone 12a to give DMT-hydroxyprolinol resorufinintermediate 26, which was then directly reacted with trimethylaceticanhydride affording the pivaloyl-protected intermediate 27. At thesecond step, the secondary hydroxy group of 27 was phosphitylated using2-cyanoethyldiisopropyl chlorophosphoramidite in the presence of atertiary amine.

In view of the methodology outlined in Reaction Scheme 12, preferred dyereagents are those having a protected phenoxazine dye attached via alinear or cyclic linking group (e.g., an alkylene, heteroalkylene orprolinol-type linker) to a phosphoramidite moiety. Most preferred is adye reagent having the formula:

In related embodiments, the present invention provides leuco phenoxazinedye reagents in which the phenoxazine portion is present in a protectedform illustrated below.

in which each of R^(x), R^(y) and R^(z) are protecting groups,preferably (C₂-C₂₀)acyl groups (e.g., acetyl, propionyl, pivaloyl,t-butoxycarbonyl, and the like) and the remaining features are asdescribed for the phenoxazine dye reagents above.

In another group of preferred embodiments, the dye reagents are xanthenedye reagents and are represented by the formula:

wherein R^(x) and R^(y) are independently selected protecting groups;R^(1′), R¹, R², R³ and R⁴ are each independently selected from the groupconsisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio,(C₁-C₈)alkoxy, aryl and heteroaryl; each R⁵ and R⁶ is independentlyselected from the group consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl,aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; wherein the alkyl portionsof any of R^(1′) and R¹ through R⁶ are optionally substituted withhalogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano,haloacetyl or hydroxy, and the alkyl portions of the substituents havefrom 1 to 6 carbon atoms; and the aryl portions of any of R^(1′) and R¹through R⁶ are optionally substituted with from one to four substituentsselected from the group consisting of halogen, cyano, carboxy, sulfo,hydroxy, amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl,(C₁-C₆)alkylthio and (C₁-C₆)alkoxy; optionally, R² taken together withR³ form a fused aromatic or heteroaromatic ring that is optionallysubstituted with from one to four substituents selected from halogencyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; and tautomeric formsthereof. The letters W, K, Y and L, as well as the subscripts p and nhave the meaning provided above with reference to the dye reagents intheir general interpretation.

Reaction Scheme 13 illustrates the preparation of a xanthene-containingphosphoramidite reagents. One of skill in the art will appreciate thatcompound 18d can be replaced by other lactone dyes in the xantheneseries (e.g., xanthene, benzo[a]xanthene, benzo[b]xanthene,benzo[c]xanthene) and the linking group (5-aminopentanol) can besubstituted with other linear or cyclic linking groups are describedherein and in the examples. Use of such substitutions provides access tothe full scope of phosphoramidite reagents contemplated by the presentinvention.

Xanthene-containing phosphoramidite 43 was prepared similarly to amidite28 starting from lactone 18d. However, in contrast to the syntheticroute in Reaction Scheme 12, the primary hydroxy group of 39 was blockedwith a transitory protecting DMT group to provide 40 and avoid acompetitive reaction with trimethylacetic anhydride during theprotection of phenol groups (see compound 41). The DMT group was readilyremoved from 41 using dilute trifluoroacetic acid solution in 10%methanol in dichloromethane. The resultant dipivaloyl intermediate 42was converted into phosphoramidite 43 using standard procedures.

Within the xanthene-based dye reagents, the dye reagent is mostpreferably one selected from the formulae:

Other preferred reagents are those wherein PA-1 or PA-2 are attached toeach of the following dyes:

wherein the dyes are shown without protecting groups (e.g., pivaloylesters) and the wavy line indicates the point of attachment to PA-1 andPA-2.

More generally, one representation of preferred phosphoramidite reagentsis provided as:

wherein R^(A), R^(B), R¹, R^(1′) and R^(x) have the meanings providedabove and R is a (C₂-C₁₂)alkylene or (C₅-C₁₂)heteroalkylene linkinggroup.

In related embodiments, 5,6-dichlorofluorescein dye reagents areprovided. More specifically, these reagents have the formula:

wherein R¹, R², R³ and R⁴ represent substituents as described above,particularly with reference to formula Ic; R^(z) represents asubstituent selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy; with theadditional feature that at least of R^(z), R¹, R², R³ and R⁴ is alinking group.

The present invention further provides oligonucleotide probes havingattached dye reagents, preferably 5,6-dichlorofluorescein dye reagentsas described above. In some embodiments, the oligonucleotide probe willfurther comprise an attached quencher for use in assays such as linearbeacon assays. In still other embodiments, the oligonucleotide probewill have an attached minor groove binder. A variety of quenchers andminor groove binders are known in the art along with methods for theirattachment to oligonucleotide probes (see, for example, co-pendingapplication Ser. Nos. 09/447,936; 09/457,616; and 09/876,830). In themost preferred embodiments, the quencher and minor groove bindercomponents are attached to the 5′-end of the oligonucleotide probe withthe 5,6-dichlorofluorescein dye attached at the 3′-end; oralternatively, the quencher and minor groove binder components areattached to the 3′-end of the oligonucleotide probe with the5,6-dichlorofluorescein dye attached at the 5′-end.

Labeled Modified Bases

The present invention further provides modified bases that are labeledusing the fused-lactone dyes described above. A variety of modifiedbases can be used and are known to those of skill in the art.Essentially, any nucleotide base having an appended reactive functionalgroup can be used, as well as nucleotide analogs (e.g., pyrimidines,purines, pyrazolopyrimidines, deazapurines, and the like). Withoutintending any limitation on this aspect of the invention, ReactionScheme 14 illustrates the reaction of a lactone dye with each of twomodified bases. These reactions are readily adapted for all lactone dyesherein.

Examples of the reaction of a chromanone dye precursor with amineligands are shown in Reaction Scheme 14. The 3-substitutedpyrazolo[3,4,d]pyrimidines or 5-substituted pyrimidines containing anamino group can be converted to related phophoramidite reagents.

Synthetic Intermediates

Certain intermediates useful in preparing the lactone dyes are alsocontemplated as an aspect of the present invention. These intermediateshave the formula:

In these formulae, R is an ester group (e.g., lower alkyl, aryl,arylalkyl, and the like); R¹, R^(a), R^(b) and R^(c)=H, hydroxy,alkyloxy, aryloxy, alkyl, aryl, substituted aryl, heterocyclylheterocyclic aromatic ring, sulfonyl, sulfamido, amino, amido, halogen,trifluoromethyl, halomethyl, or optionally two adjacent members of R¹,R^(a), R^(b) and R^(c) are combined to form a 5-7 membered ring; and R⁵,R⁶, R⁷, R⁸=H, alkyl, aryl, arylalkyl or optionally, two of R⁵, R⁶, R⁷and R⁸ are combined to form a 5-7 membered ring, with the proviso thatat least one of R^(a) or R^(c) is hydroxy, and at least one of R¹ orR^(b) is hydrogen.

Preferably, R¹ and R^(a), or R^(a) and R^(b) or R^(b) and R^(c) form anaromatic or non-aromatic ring, or a heterocyclic structure.

In other preferred embodiments, R⁵ through R⁸ are H, and R^(a) is OH.More preferably, R^(c) is H and R is methyl or ethyl. Still morepreferably, R¹ is Cl, F or phenyl (see below).

EXAMPLES

Instrumentation: HPLC system equipped with photo diode array (PDA)detector. Column-Rainin C18, Dynamax-100A, SHORT-ONE (4.6×100 mm).Mobile phase was a gradient of acetonitrile (0-100%) in 0.1 Mtriethylammonium acetate (pH 7.5) in 10 min followed by 10 min 100%acetonitrile wash. Flow rate was 1 ml/min. Sample loop was 20 μl.

Example 1

3-Chloro-2,4-dimethoxybenzaldehyde (2a) was prepared from1,3-dimethoxybenzene (1a) according to Plattner, J. J. et al. J. Med.Chem. 1984, 27 (8), 1016-1026.

3-(3-Chloro-2,4-dimethoxyphenyl)acrylic acid (3a). A solution ofcompound (2a) (11.07 g, 55.2 mmol), malonic acid (8.639 g, 83 mmol) andpiperidine (2 ml, 1.722 g, 20.2 mmol) in 80 ml of anhydrous pyridine wasrefluxed (bath temperature 110° C.) for 2 h. Reaction mixture wascooled, concentrated under vacuum and acidified 250 ml of 5% aqueouscitric acid to a pH of 3. Resulted heterogeneous mixture was sonicatedfor a few minutes. The precipitated material was filtered off, washedwith water (2×20 ml) and dried in vacuum over KOH to give 12.7 g (52.3mmol, 95% yield) of pure (HPLC, ¹H NMR) desired acid 3a as a whitesolid. ¹H NMR (DMSO-d₆): δ 7.77 (d, J=9.0 Hz, 1H), 7.67 (d, J=16.0 Hz,1H), 7.01 (d, J=9.0 Hz, 1H), 6.51 (d, J=16.0 Hz, 1H), 3.90 (s, 3H), 3.77(s, 3H).

3-(3-Chloro-2,4-dimethoxyphenyl)propionic acid (4a). A suspension of 3a(12.57 g, 51.88 mmol) in a mixture of MeOH (50 ml) and THF (200 ml) washydrogenated at 50 psi in the presence of 10% Pd/C (0.3 g) for 4 h. Thecatalyst was removed by filtration through Celite and the filtrate wasconcentrated to afford 12.6 g (51.5 mmol, 99% yield) of analyticallypure (HPLC, ¹H NMR) 4a as a white solid. ¹H NMR (DMSO-d₆): δ 7.15 (d,J=8.6 Hz, 1H), 6.86 (d, J=8.6 Hz, 1H), 3.81 (s, 3H), 3.75 (s, 3H), 2.78(t, J=7.5 Hz, 2H), 2.49 (t, J=7.5 Hz, 2H).

Methyl 3-(3-chloro-2,4-dihydroxyphenyl)propanoate (5a). A solution of 1(10.0 g, 40.9 mmol) in a mixture of acetic acid (75 ml, 78.67 g, 1.3mol) and 48% aqueous hydrobromic acid (75 ml, 111.75 g, 0.66 mol) wasrefluxed (bath temperature +120° C.) for 15 h. Reaction mixture wascooled and concentrated under vacuum. The obtained solid material wasdried by co-evaporation with toluene (3×100 ml) and dissolved in 100 mlof methanol. Hydrogen chloride was bubbled for 5 min. The resultant hotsolution was allowed to cool to room temperature and then concentrated.The crude product was chromatographed on silica gel eluting with 1:4EtOAc-hexane. Concentration of the pure product fractions afforded 9.2 g(98%) of the title compound as a pale tan oil, which slowly solidifiedduring drying in vacuo. ¹H NMR (DMSO-d₆): δ 9.80 (s, 1H), 8.95 (s, 1H),6.81 (d, J=8.4 Hz, 1H), 6.38 (d, J=8.4 Hz, 1H), 3.57 (s, 3H), 2.73 (t,J=7.5 Hz, 2H), 2.50 (t, J=7.5 Hz, 2H).

Compounds 2b, 3b, 4b and 5b were synthesized in similar yields using theprocedures described for corresponding chloroanalogs.

3-Fluoro-2,4-dimethoxybenzaldehyde (2b). ¹H NMR (DMSO-d₆): δ 10.11 (s,1H), 7.56 (dd, J=9 Hz, J=2 Hz, 1H), 7.08 (t, J=8 Hz, 1H), 4.00 (d,J=2,3H), 3.93 (s, 3H).

(2E)-3-(3-Fluoro-2,4-dimethoxyphenyl)prop-2-enoic acid (3b). ¹H NMR(DMSO-d₆): δ 12.36 (s, 1H), 7.66 (d, J=16 Hz, 1H), 7.56 (d, J=8.5 Hz,1H), 6.96 (t, J=8.5 Hz, 1H), 6.57 (d, J=16 Hz, 1H), 3.89 (s, 3H), 3.87(s, 3H).

3-(3-Fluoro-2,4-dimethoxyphenyl)propanoic acid (4b). ¹H NMR (DMSO-d₆): δ11.8 (br s, 1H), 6.93 (d, J=8.5 Hz, 1H), 6.80 (t, J=8.5 Hz, 1H), 3.83(s, 3H), 3.79 (s, 3H), 2.74 (t, J=7.5 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H).

Methyl 3-(3-fluoro-2,4-dihydroxyphenyl)propanoate (5b). ¹H NMR(DMSO-d₆): δ 9.47 (s, 1H), 9.33 (s, 1H), 6.62 (d, J=8.5 Hz, 1H), 6.29(t, J=8.5 Hz, 1H), 3.56 (s, 3H), 2.70 (t, J=7.5 Hz, 2H), 2.49 (t, J=7.5Hz, 2H). 2,4-Dimethoxy-3-phenylbenzaldehyde (2c). Compound 2c wassynthesized from 1c in 95% yield using the procedure described for 2a.¹H NMR(acetone-d₆): δ 10.28 (s, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.42-7.49(m, 5H), 7.10 (d, J=8.8 Hz, 1H), 3.89 (s, 3H), 3.48 (s, 3H).

(2E)-3-(2,4-Dimethoxy-3-phenylphenyl)prop-2-enoic acid (3c). Compound 3cwas synthesized from 2c in 96% yield using the procedure described for3a. ¹H NMR (DMSO-d₆): δ 7.81 (d, J=8.8 Hz, 1H), 7.74 (d, J=16.0 Hz, 1H),7.28-7.42 (m, 5H), 6.97 (d, J=8.8 Hz, 1H), 6.47 (d, J=16.0 Hz, 1H), 3.73(s, 3H), 3.25 (s, 3H).

3-(2,4-Dimethoxy-3-phenylphenyl)propanoic acid (4c). Compound 4c wassynthesized from 3c in 98% yield using the procedure described for 4a.¹H NMR (DMSO-d₆): δ 12.0 (br s, 1H), 7.39 (m, 2H), 7.29 (m, 3H), 7.18(d, J=8.5 Hz, 1H), 6.80 (d, J=8.5 Hz, 1H), 3.64 (s, 3H), 3.18 (s, 3H),2.78 (t, J=7.5 Hz, 2H), 2.50 (t, J=7.5 Hz, 2H). Methyl3-(2,4-dihydroxy-3-phenylphenyl)propanoate (5c). BBr₃ (4.72 g, 1.78 ml,18.9 mmol) was carefully added through a syringe over 5 min to a stirredsolution of dimethoxy acid 4c (1.35 g, 4.7 mmol) in anhydrous CH₂Cl₂ (20ml). Resulted mixture was magnetically stirred for 8 h and concentratedunder vacuum. The residue was co-evaporated with toluene (2×20 ml),dissolved in methanol (20 ml) and HCl gas was bubbled into the solutionfor 2 min. The material obtained after evaporation of the solvent waspartitioned between cold water (30 ml) and ethyl acetate (100 ml). Theorganic phase was washed with saturated NaHCO₃ (2×10 ml), brine (20 ml),dried over MgSO₄ and concentrated. The resultant crude product waschromatographed on a short silica gel column eluting with ethyl acetate.Pure product fractions were concentrated to afford 1.2 g (94%) of 5c asa tan oil. ¹H NMR(DMSO-d₆): δ 8.95 (s, 1H), 7.82 (s, 1H), 7.24-7.36 (m,5H), 6.83 (d, J=8.2 Hz, 1H), 6.38 (d, J=8.2 Hz, 1H), 3.59 (s, 3H), 2.75(t, J=8.0 Hz, 2H), 2.52 (t, J=8.0 Hz, 2H).

Methyl 3-(8-chloro-7-hydroxy-4-methyl-2-oxo-2H-chromen-6-yl)propanoate(6a). Diketene (0.7 ml, 9.1 mmol) was added dropwise to a stirredsolution of 5a (1.0 g, 4.3 mmol) in 7 ml of methanesulfonic acid. Theresultant solution was heated at 70° C. for 5 min and cooled. Methanol(20 ml) was added and the reaction was heated to boiling; this treatmentesterified small amount of free acid formed during the cyclization step.Methanol was evaporated and water was added to precipitate the product.The crude material was chromatographed on silica eluting with ethylacetate. Concentration of the pure product fractions and drying undervacuum afforded 0.82 g (64%) of the desired product as a pale yellowsolid. ¹H NMR (DMSO-d₆): δ 10.45 (s, 1H), 7.50 (s, 1H), 6.24 (s, 1H),3.59 (s, 3H), 2.93 (t, J=7.5 Hz, 2H), 2.62 (t, J=7.5 Hz, 2H), 2.38 (s,3H).

3-(8-Chloro-7-hydroxy-4-methyl-2-oxo-2H-chromen-6-yl)propanoic acid(7a). Ester 6a (0.75 g, 2.5 mmol) was dissolved in 10 ml of 1N NaOH.After being kept at room temperature for 30 min the solution wasacidified with 1N HCl to pH of 3. The precipitated material wascollected by filtration and washed with water. Drying under vacuum overP₂O₅ afforded 0.705 g of acid 7a as an off-white solid. ¹H NMR(DMSO-d₆): δ 12.2 (br s, 1H), 10.41 (br s, 1H), 7.50 (s, 1H), 6.23 (s,1H), 2.89 (t, J=7.5 Hz, 2H), 2.53 (t, J=7.5 Hz, 2H), 2.38 (s, 3H).

10-Chloro-6-methyl-2H-pyrano[5,6-g]chromane-2,8-dione (8a).Pentafluorophenyl trifluoroacetate (PFP-TFA) (0.4 ml, 2.3 mmol) wasadded to a solution of 7a (0.65 g, 2.3 mmol) and triethylamine (0.5 ml)in 8 ml of anhydrous CH₂Cl₂ and the reaction was stirred for 1 h. Theprecipitated product was filtered off and washed with ethyl acetate.Drying under vacuum afforded 0.5 g (82%) of the desired lactone as apale yellow solid. ¹H NMR (DMSO-d₆): δ 7.73 (s, 1H), 6.43 (s, 1H), 3.13(t, J=7.5 Hz, 2H), 2.89 (t, J=7.5, 2H), 2.43 (s, 3H).

10-Fluoro-6-methyl-2H-pyrano[5,6-g]chromane-2,8-dione (8b) wassynthesized from compound 5b by analogy with chloro analog 8a withsimilar chemical yield.

Methyl 3-(8-fluoro-7-hydroxy-4-methyl-2-oxo-2H-chromen-6-yl)propanoate(6b). ¹H NMR (DMSO-d₆): δ 10.85 (s, 1H), 7.33 (s, 1H), 6.21 (s, 1H),3.59 (s, 3H), 2.89 (t, J=7.5 Hz, 2H), 2.62 (t, J=7.5 Hz, 2H), 2.36 (s,3H).

3-(8-Fluoro-7-hydroxy-4-methyl-2-oxo-2H-chromen-6-yl)propanoic acid(7b). ¹H NMR (DMSO-d₆): δ 12.2 (br s, 1H), 10.81 (br s, 1H), 7.33 (s,1H), 6.21 (s, 1H), 2.88 (t, J=7.5 Hz, 2H), 2.52 (t, J=7.5 Hz, 2H), 2.36(s, 3H).

10-Fluoro-6-methyl-2H-pyrano[5,6-g]chromane-2,8-dione (8b). ¹H NMR(DMSO-d₆): δ 7.56 (s, 1H), 6.42 (s, 1H), 3.13 (t, J=7.5 Hz, 2H), 2.89(t, J=7.5, 2H), 2.41 (s, 3H).

Compounds 9a, 9b, 9c were synthesized by the general procedure describedfor 4-nitrosoresorcinol (Kendall et. al. J. Amer. Chem. Soc. 82: 1853-4(1960)).

Methyl 3-(7-hydroxy-10-oxido-3-oxo-3H-phenoxazin-8-yl)propanoate (10a).4-Nitrosoresorcinol (9a) (33.13 g, 238 mmol), synthesized by theprocedure of Kendall et. al. J. Amer. Chem. Soc. 82: 1853-4 (1960) wasdissolved in 1.1 L of methanol at +40° C. with sonication. Resultantsolution was transferred into a 3 L round bottom flask and cooled to +2°C. using an ice-water bath. A solution of ester 5d (46.8 g, 238 mmol) inmethanol (100 ml) and MnO₂ (20.68 g, 238 mmol) were added and themixture was allowed to cool to 0-2° C. Concentrated sulfuric acid (25.5ml, 46.9 g, 478 mmol) was added over 2 min with cooling and intensivestirring. The resultant mixture was stirred at room temperature for 4 hand then diluted with ether (1.2 L). Precipitated material was collectedby filtration, washed with MeOH/ether (1:1) mixture (2×100 ml) and ether(2×100 ml). Drying under vacuum afforded 26.32 g of 10a (35%) as ablack-brown solid, m.p. 233° C. (dec.). ¹H NMR (DMSO-d₆): δ 11.24 (bs,1H), 8.00 (d, J=9.1 Hz, 1H), 7.83 (s, 1H), 6.88 (br d, J=9.1 Hz, 1H),6.82 (br s, 1H), 6.20 (b s, 1H), 3.59 (s, 3H), 2.75 (t, J=7.3 Hz, 2H),2.59 (t, J=7.3 Hz, 2H).

Compounds 10b, 10c, 10d, 10e were prepared by analogy with thenon-halogenated analog 10a.

Methyl3-(6-chloro-7-hydroxy-10-oxido-3-oxo-3H-phenoxazin-8-yl)propanoate(10b). ¹H NMR (DMSO-d₆): δ 11.4 (br s, 1H), 8.05 (d, J=7 Hz, 1H), 7.87(s, 1H), 6.88 (m, 2H), 3.59 (s, 3H), 2.80 (t, J=7.5 Hz, 2H), 2.61 (t,J=7.5 Hz, 2H).

Methyl3-(6-fluoro-7-hydroxy-10-oxido-3-oxo-3H-phenoxazin-8-yl)propanoate(10c). ¹H NMR (DMSO-d₆): δ 11.35 (br s, 1H), 8.05 (d, J=7 Hz, 1H), 7.81(s, 1H), 6.95 (m, 2H), 3.59 (s, 3H), 2.79 (t, J=7.5 Hz, 2H), 2.61 (t,J=7.5 Hz, 2H).

Methyl3-(6-fluoro-7-hydroxy-10-oxido-3-oxo-3H-phenoxazin-8-yl)propanoate(10d). ¹H NMR (DMSO-d₆): δ 11.80 (br s, 1H), 7.86 (d, J=9 Hz, 1H), 7.83(s, 1H), 7.07 (t, J=9 Hz, 1H), 3.59 (s, 3H), 2.79 (t, J=7.5 Hz, 2H),2.62 (t, J=7.5 Hz, 2H).

3-(7-Hydroxy-3-oxo-3H-phenoxazin-8-yl)propanoic acid (11a). Methyl ester(10a) (22.83 g, 72.4 mmol) was dissolved in 400 ml of 0.5 M aqueoussodium hydroxide and the solution was stirred for 1 h. A solution ofsodium dithionite solution (200 ml, 100 mmol) was added and reactionmixture was stirred for additional 15 minutes. To precipitate theproduct 3 M hydrochloric acid (80 ml) was carefully added and resultantsuspension was stirred for 10 min and centrifuged. The supernatant wasdecanted and the residue was re-suspended in ˜800 ml of water andcentrifiged again. This operation was repeated one more time. Finalsupernatant had pH of 3-4. The centrifuged material was dried undervacuum overnight at 40° C. to afford 14.12 g (68%) of desired product(11a) as a brown solid. ¹H NMR (CD₃OD, +40° C.): δ 7.64 (d, J=9.0 Hz,1H), 7.39 (s, 1H), 6.89 (d, J=9.0 Hz, 1H), 6.74 (s, 1H), 6.34 (s, 1H),2.63 (m, 2H), 2.51 (m, 2H).

Compounds 11b, 11c, 11d, 11e were prepared by analogy with thenon-halogenated analog 11a.

3-(4-Chloro-7-hydroxy-3-oxophenoxazin-2-yl)propanoic acid (11b). ¹H NMR(DMSO-d₆): δ 12.2 (br s, 1H), 11.2 (br s, 1H), 7.74 (d, J=9 Hz, 1H),7.39 (s, 1H), 6.96 (d, J=9 Hz, 1H), 6.91 (s, 1H), 2.76 (t, J=7.5 Hz,2H), 2.56 (t, J=7.5 Hz, 2H).

3-(4-Fluoro-7-hydroxy-3-oxophenoxazin-2-yl)propanoic acid (11c). ¹H NMR(DMSO-d₆): δ 12.2 (br s, 1H), 11.2 (br s, 1H), 7.70 (d, J=8.5 Hz, 1H),7.34 (s, 1H), 6.92 (d, J=8.5 Hz, 1H), 6.90 (s, 1H), 2.74 (t, J=7.5 Hz,2H), 2.53 (t, J=7.5 Hz, 2H).

3-(4,6-Difluoro-7-hydroxy-3-oxophenoxazin-2-yl)propanoic acid (11d). ¹HNMR (DMSO-d₆): δ 12.2 (br s, 1H), 11.6 (br s, 1H), 7.54 (d, J=8.5 Hz,1H), 7.35 (s, 1H), 6.92 (t, J=8.5 Hz, 1H), 2.75 (t, J=7.5 Hz, 2H), 2.54(t, J=7.5 Hz, 2H).

3-(4,6,8-Trifluoro-7-hydroxy-3-oxophenoxazin-2-yl)propanoic acid (11e).¹H NMR (DMSO-d₆): δ 12.2 (br s, 1H), 11.6 (br s, 1H), 7.65 (d, J=10 Hz,1H), 7.44 (s, 1H), 2.83 (t, J=7.5 Hz, 2H), 2.56 (t, J=7.5 Hz, 2H).

3,4-dihydro-2H-pyrano[3,2-b]phenoxazine-2,9-dione(12a).

To a suspension of acid 12a (10.36 g, 36.3 mmol) in anhydrous DMF (200ml) was added N,N,-diisopropylethylamine (26.13 ml, 150 mmol) andresultant mixture was magnetically stirred for 10 min. The reactionflask was placed in water bath (t-+20° C.) and PFP-TFA (18.9 ml, 110mmol) was added through a syringe during 5-10 min. After being stirredfor 2 h, the reaction mixture was concentrated under vacuum and dilutedwith ether (200 ml). The precipitated material was filtered off andwashed with ether (2×30 ml), 2-propanol (2×50 ml) and ether (3×30 ml).Drying under vacuum afforded 6.56 g (67%) of the desired lactone 12a asan orange solid. m.p. 268° C. (dec.). ¹H NMR (DMSO-d₆, +45° C.): δ 7.81(s, 1H), 7.54 (d, J=9.8 Hz, 1H), 7.27 (s, 1H), 6.82 (dd, J=9.8 Hz, J=2.1Hz, 1H), 6.29 (d, J=2.1 Hz, 1H), 3.12 (m, 2H), 2.88 (m, 2H).

Compounds 12b, 12c, 12d, 12e were prepared by analogy with thenon-halogenated analog 12a.

12-Chloro-3,4-dihydro-2H-pyrano[3,2-b]phenoxazine-2,9-dione (12b). ¹HNMR (DMSO-d₆, +45° C.): δ 7.80 (s, 1H), 7.57 (d, J=9.8 Hz, 1H), 6.85 (d,J=9.8 Hz, 1H), 6.39 (s, 1H), 3.16 (t, J=7.5 Hz, 2H), 2.91 (t, J=7.5 Hz,2H).

12-Fluoro-3,4-dihydro-2H-pyrano[3,2-b]phenoxazine-2,9-dione (12c). ¹HNMR (DMSO-d₆, +45° C.): δ 7.65 (s, 1H), 7.57 (d, J=9.8 Hz, 1H), 6.84 (d,J=9.8 Hz, 1H), 6.42 (s, 1H), 3.16 (t, J=7.5 Hz, 2H), 2.92 (t, J=7.5 Hz,2H).

10,12-Difluoro-3,4-dihydro-2H-pyrano[3,2-b]phenoxazine-2,9-dione (12d).¹H NMR (DMSO-d₆): δ 7.70 (s, 1H), 7.61 (d, J=9.9 Hz, 1H), 6.84 (dd,J1=9.9 Hz, J2=1 Hz, 1H), 3.18 (t, J=7.5 Hz, 2H), 2.93 (t, J=7.5 Hz, 2H).

8,10,12-Trifluoro-3,4-dihydro-2H-pyrano[3,2-b]phenoxazine-2,9-dione(12e). ¹H NMR (DMSO-d₆): δ 7.77 (s, 1H), 7.72 (d, J=10 Hz, 1H), 3.20 (t,J=7.5 Hz, 2H), 2.94 (t, J=7.5 Hz, 2H).

3,4,5,6-tetrachloro-2-({3-chloro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14a). Tetrachlorophthalic anhydride (17.4 g, 60.8 mmol) wassuspended in 160 ml of anhydrous 1,2-dichloroethane and stirred for 30min to dissolve most of the solid. AlCl₃ (21.0 g, 158 mmol) was added inone portion (light exotherm) followed by addition of ester 5a (14.0 g,60.7 mmol) as a solution in anhydrous 1,2-dichloroethane (80 ml). Themixture was stirred for 15-20 min to give a clear solution. Thenreaction mixture was stirred at room temperature for another 20 h. Bythat time the starting compounds were almost completely consumedaccording to the HPLC analysis.

The solvent was evaporated and the resulting gummy residue waspartitioned between ethyl acetate (500 ml) and cold 5 N HCl (500 ml).The aqueous phase was extracted with more ethyl acetate, the combinedorganic solution was washed with 1 N HCl (300 ml), saturated NaCl (300ml) and dried over MgSO₄. Concentration of the solution gave a paleyellow solid; it was triturated with CH₂Cl₂ (200 ml), cooled to 0° C.and then filtered off. Washing with CH₂Cl₂ (2×50 ml) and drying undervacuum afforded 21.0 g of the desired product as a white solid. Themother liquor afforded additional 2.1 g of the product. Total yield was23.1 g (74%). ¹H NMR (DMSO-d₆): δ 11.95 (bs, 1H), 11.05 (bs, 1H), 7.21(s, 1H), 3.54 (s, 3H), 2.75 (m, 2H), 2.45 (m, 2H).

Compounds 14b-14n were synthesized analogously to 14a.

3,4,5,6-Tetrafluoro-2-({3-fluoro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14b). ¹H NMR (DMSO-d₆): δ 12.0 (bs, 1H), 11.05 (bs, 1H), 7.02 (s,1H), 3.54 (s, 3H), 2.72 (t, J=7.5 Hz, 2H), 2.45 (m, 2H).

3,4,5,6-Tetrachloro-2-({3-fluoro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14c). ¹H NMR (DMSO-d₆): δ 11.3 (bs, 1H), 11.1 (bs, 1H), 7.17 (s,1H), 3.55 (s, 3H), 2.72 (m, 2H), 2.45 (m, 2H).

2-({3-Chloro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)-3,4,5,6-tetrafluorobenzoicacid (14d). ¹H NMR (DMSO-d₆): δ 11.94 (bs, 1H), 10.99 (bs, 1H), 7.19 (s,1H), 3.54 (s, 3H), 2.74 (t, J=7.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H).

4,5-Dichloro-2-({3-chloro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14e). ¹H NMR (DMSO-d₆): δ 12.46 (s, 1H), 8.16 (s, 1H), 7.87 (s,1H), 6.90 (s, 1H), 3.49 (s, 3H), 2.70 (t, J=7.3 Hz, 2H), 2.42 (t, J=7.3Hz, 2H).

4,5-Dichloro-2-({3-fluoro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl/carbonyl)benzoicacid (14f). ¹H NMR (DMSO-d₆): δ 11.59 (bs, 1H), 11.13 (bs, 1H), 8.13 (s,1H), 7.82 (s, 1H), 6.83 (s, 1H), (s, 1H), 3.50 (s, 3H), 2.67 (t, J=7.5Hz, 2H), 2.43 (t, J=7.5 Hz, 2H).

2-({2,4-Dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)-3,4,5,6-tetrafluorobenzoicacid (14g). ¹H NMR (DMSO-d₆): δ 11.2 (bs, 1H), 11.04 (bs, 1H), 7.02 (s,1H), 6.37 (s, 1H), 3.54 (s, 3H), 2.65 (t, J=7.5 Hz, 2H), 2.46 (t, J=7.5Hz, 2H).

2-({3-Chloro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14h). ¹H NMR (DMSO-d₆): δ 11.75 (bs, 1H), 8.02 (d, J=7.2 Hz, 1H),7.68 (m, 2H), 7.44 (d, J=7.2 Hz, 1H), 6.75 (s, 1H), 3.45 (s, 3H), 2.65(t, J=7.5 Hz, 2H), 2.39 (t, J=7.5 Hz, 2H).2-((2,4-Dihydroxy-5-[2-(methoxycarbonyl)ethyl]-3-phenylphenyl}carbonyl)benzoicacid (14i). ¹H NMR (DMSO-d₆): δ 7.95 (m, 1H), 7.25-7.55 (m, 9H), 6.75(s, 1H), 2.62 (t, J=7.4 Hz, 2H), 2.39 (t, J=7.3 Hz, 2H).

2-({2,4-Dihydroxy-5-[2-(methoxycarbonyl)ethyl]-3-phenylphenyl-carbonyl)-4,5-dichlorobenzoicacid (14j). ¹H NMR (DMSO-d₆): δ 7.36-7.41 (m, 2H), 7.32-7.20 (m, 3H),7.11 (bs, 1H), 3.54 (s, 3H), 2.73 (m, 2H), 2.49 (m, 2H).

2-({2,4-Dihydroxy-5-[2-(methoxycarbonyl)ethyl]-3-phenylphenyl}carbonyl)-3,4,5,6-tetrachlorobenzoicacid (14k). ¹H NMR (DMSO-d₆): δ 12.25 (s, 1H), 9.55 (s, 1H), 8.17 (s,1H), 7.88 (s, 1H), 7.20-7.47 (m, 5H), 6.90 (s, 1H), 3.50 (s, 3H), 2.70(t, J=7.0 Hz, 2H), 2.43 (t, J=7.1 Hz, 2H).

2-({2,4-Dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14l). ¹H NMR (DMSO-d₆): δ 12.14 (s, 1H), 10.89 (s, 1H), 8.00 (d,J=7.2 Hz, 1H), 7.62 (m, 2H), 7.39 (d, J=7.2 Hz, 1H), 6.76 (s, 1H), 6.37(s, 1H), 3.45 (s, 3H), 2.56 (t, J=7.5 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H).

2-({2,4-Dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)-4,5-dichlorobenzoicacid (14m). ¹H NMR (DMSO-d₆): δ 11.71 (s, 1H), 10.93 (s, 1H), 8.12 (s,1H), 7.77 (s, 1H), 6.92 (s, 1H), 6.37 (s, 1H), 3.49 (s, 3H), 2.60 (t,J=7.5 Hz, 2H), 2.41 (t, J=7.5 Hz, 2H).

2-({3-Fluoro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14n). ¹H NMR (DMSO-d₆): δ 12.03 (s, 1H), 11.12 (s, 1H), 8.00 (d,J=7.2 Hz, 1H), 7.62 (m, 2H), 7.42 (d, J=7.2 Hz, 1H), 6.64 (s, 1H), 3.45(s, 3H), 2.63 (t, J=7.5 Hz, 2H), 2.40 (t, J=7.5 Hz, 2H).

3,6-Dichloro-2-({3-chloro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14o). ¹H NMR (DMSO-d₆): δ 12.15 (s, 1H), 11.00 (s, 1H), 7.78 (s,2H), 6.97 (s, 1H), 3.51 (s, 3H), 2.73 (t, J=7.5 Hz, 2H), 2.44 (t, J=7.5Hz, 2H).

3,6-Difluoro-2-({3-chloro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (14p). ¹H NMR (DMSO-d₆): δ 12.19 (br s, 1H), 10.95 (br s, 1H), 7.62(m, 2H), 7.04 (s, 1H), 3.50 (s, 3H), 2.72 (t, J=7.5 Hz, 2H), 2.44 (t,J=7.5 Hz, 2H).

Methyl 3-(4,5,6,7,13,16-hexachloro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoate(16a). 4-Chlororesorcinol (15a) (0.56 g, 3.9 mmol) and 14a (1.0 g, 1.93mmol) were suspended in 10 ml of methanesulfonic acid. The suspensionwas stirred at 70° C. under argon for 1 h and cooled. A mixture of iceand water (50 g) was added to precipitate the product. Ethyl acetate (50ml) was added to extract the precipitate. The product from organic layerwas extracted with sodium bicarbonate solution. The aqueous phase waswashed with ether to remove excess 4-chlororesorcinol and acidified withconcentrated hydrochloric acid. The precipitated material, pure product16a, was taken up in ethyl acetate and dried over Na₂SO₄. Concentrationof the solution and drying under vacuum afforded 1.18 g (97%) of thetitle compound 16a as an orange solid. ¹H NMR (DMSO-d₆): δ 11.20 (s,1H), 10.17 (s, 1H), 7.37 (s, 1H), 6.91 (s, 1H), 6.82 (s, 1H), 3.47 (s,3H), 2.75 (t, J=7.2 Hz, 2H), 2.45 (t, J=7.2 Hz, 2H).

Compounds 16b-16g were synthesized from corresponding intermediates 14and 15 using procedure described for 16a.

Methyl3-(16-chloro-4,5,6,7,13-pentafluoro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoate(16b). ¹H NMR (DMSO-d₆): δ 11.27 (s, 1H), 10.64 (s, 1H), 7.37 (s, 1H),6.96 (s, 1H), 6.75 (s, 1H), 3.50 (s, 3H), 2.71 (t, J=7.2 Hz, 2H), 2.49(t, J=7.2 Hz, 2H).

Methyl 3-(4,5,6,7,16-pentachloro-13-fluoro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoate(16c). ¹H NMR (DMSO-d₆): δ 11.23 (s, 1H), 10.55 (s, 1H), 7.35 (s, 1H),6.96 (s, 1H), 6.66 (s, 1H), 3.46 (s, 3H), 2.71 (t, J=7.2 Hz, 2H), 2.43(t, J=7.2 Hz, 2H).

Methyl3-(13,16-dichloro-4,5,6,7-tetrafluoro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoate(16d). ¹H NMR (DMSO-d₆): δ 11.28 (bs, 1H), 10.27 (bs, 1H), 7.38 (s, 1H),6.95 (s, 1H), 6.91 (s, 1H), 3.50 (s, 3H), 2.76 (t, J=7.2 Hz, 2H), 2.49(t, J=7.2 Hz, 2H).

Methyl3-(16-chloro-4,5,6,7-tetrafluoro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoate(16e). ¹H NMR (DMSO-d₆): δ 11.75 (s, 1H), 10.48 (s, 1H), 7.31 (s, 1H),6.90 (s, 1H), 6.84 (s, 1H), 6.73 (s, 1H), 3.50 (s, 3H), 2.67 (t, J=7.5Hz, 2H), 2.45 (t, J=7.5 Hz, 2H).

Methyl3-(4,5,6,7-tetrafluoro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoate(16f). ¹H NMR (DMSO-d₆): δ 10.42 (s, 1H), 10.27 (s, 1H), 6.98 (d, J=9Hz, 1H), 6.86 (s, 1H), 6.72 (s, 1H), 6.69 (d, J=2 Hz, 1H), 6.57 (dd,J₁=9 Hz, J₂=2 Hz, 1H), 3.50 (s, 3H), 2.68 (t, J=7.5 Hz, 2H), 2.47 (t,J=7.5 Hz, 2H).

Methyl3-(4,5,6,7,16-pentachloro-12,15-dihydroxy-1-oxo-13-phenylspiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoate(16g). ¹H NMR (DMSO-d₆): δ 10.90 (s, 1H), 8.92 (s, 1H), 7.40-7.53 (m,5H), 7.32 (s, 1H), 6.79 (s, 1H), 6.50 (s, 1H), 3.49 (s, 3H), 2.76 (t,J=7.4 Hz, 2H), 2.50 (t, J=7.4 Hz, 2H).

3-(4,5,6,7,13,16-Hexachloro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17a). A suspension of 14a (20.0 g, 39 mmol) and 4-chlororesorcinol(15a) (20.0 g, 138 mmol) in 200 ml of trifluoroacetic acid was heated at70° C. with stirring for 5 min to dissolve the solids. Methanesulfonicacid (10 ml) was added in one portion and the reaction was stirred at70° C. for 2 h. The mixture was cooled and concentrated under vacuum.The oily residue was triturated with water (500 ml) and extracted withethyl acetate (300 ml). The organic phase was washed with 5% sodiumbicarbonate solution to extract the product; the aqueous phase waswashed with ether and then acidified with hydrochloric acid to pH of 3.The fine precipitate was collected by filtration on sintered glassfunnel and washed with water. Drying under vacuum (over P₂O₅) afforded21.6 g (90%) of the title acid 17a as an orange solid. ¹H NMR (DMSO-d₆):δ 11.20 (s, 1H), 10.16 (s, 1H), 7.37 (s, 1H), 6.91 (s, 1H), 6.82 (s,1H), 2.75 (t, J=7.5 Hz, 2H), 2.45 (t, J=7.5 Hz, 2H).

3-(13,16-Dichloro-4,5,6,7-tetrafluoro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17b). ¹H NMR (DMSO-d₆): δ 11.95 (br s, 1H), 11.27 (br s, 1H),10.21 (br s, 1H), 7.37 (s, 1H), 7.21 (s, 1H), 6.93 (s, 1H), 2.70 (t,J=7.5 Hz, 2H), 2.35 (t, J=7.5 Hz, 2H).

3-(12,15-Dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17c). ¹H NMR (DMSO-d₆): δ 10.3 (br s, 1H), 10.2 (br s, 1H), 8.00(d, J=7.2 Hz, 1H), 7.75 (m, 2H), 7.23 (d, J=7.2 Hz, 1H), 6.71 (s, 1H),6.66 (s, 1H), 6.51 (m, 2H), 6.44 (s, 1H), 2.57 (t, J=7.5 Hz, 2H), 2.30(t, J=7.5 Hz, 2H).

3-(4,7,13,16-Tetrachloro-12,15-dihydroxy-14-methyl-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17d). ¹H NMR (DMSO-d₆): δ 12.02 (br s, 1H), 10.09 (s, 1H), 9.96(s, 1H), 7.81 (s, 2H), 6.97 (s, 1H), 6.75 (s, 1H), 2.69 (m, 2H), 238 (s,3H), 2.35 (t, J=7.5 Hz, 2H).

3-(4,7,13,16-Tetrachloro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9-xanthene]-11-yl)propanoicacid (17e). ¹H NMR (DMSO-d₆): δ 12.07 (brs, 1H), 11.14 (brs, 1H), 10.02(br s, 1H), 7.79 (s, 2H), 7.14 (s, 1H), 6.91 (s, 1H), 6.75 (s, 1H), 2.70(m, 2H), 2.34 (t, J=7.5 Hz, 2H).

3-(4,7,13-Trichloro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17f). ¹H NMR (DMSO-d₆): δ 12.08 (br s, 1H), 10.24 (br s, 1H),10.02 (br s, 1H), 7.81 (s, 2H), 6.80 (s, 1H), 6.77 (d, J=9 Hz, 1H), 6.70(d, J=2 Hz, 1H), 6.59 (dd, J₁=9 Hz, J₂=2 Hz, 1H), 2.71 (m, 2H), 2.35 (t,J=7.5 Hz, 2H).

3-(13,16-Dichloro-4,7-difluoro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17g). ¹H NMR (DMSO-d₆): δ 12.05 (br s, 1H), 11.19 (s, 1H), 10.11(s, 1H), 7.64 (m, 2H), 7.24 (s, 1H), 6.93 (s, 1H), 6.86 (s, 1H), 2.70(m, 2H), 3.36 (t, J=7.5 Hz, 2H).

3-(5,6,13,16-Tetrachloro-12,15-dihydroxy-14-methyl-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17h). ¹H NMR (DMSO-d₆): δ 12.08 (br s, 1H), 10.05 (br s, 1H), 9.98(br s, 1H), 8.27 (s, 1H), 7.81 (s, 1H), 6.81 (s, 1H), 6.66 (s, 1H), 2.66(m, 2H), 2.39 (s, 3H), 2.34 (m, 2H).

3-(5,6,13,16-Tetrachloro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoicacid (17i). ¹H NMR (DMSO-d₆): δ 12.06 (br s, 1H), 11.14 (br s, 1H),10.08 (br s, 1H), 8.26 (s, 1H), 7.81 (s, 1H), 6.97 (s, 1H), 6.91 (s,1H), 6.65 (s, 1H), 2.67 (m, 2H), 2.34 (m, 2H).

3-(5,6,13-Trichloro-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′xanthene]-11-yl)propanoicacid (17j). ¹HNMR(DMSO-d₆): δ 12.07 (brs, 1H), 11.14 (brs, 1H), 10.05(br s, 1H), 8.29 (s, 1H), 7.80 (s, 1H), 6.67 (m, 4H), 2.66 (m, 2H), 2.34(m, 2H).

8,12,22,23,24,25-Hexachloro-9-hydroxyspiro[3,4-dihydro-2H-pyrano[3,2-b]xanthene-6,3′-3-hydroisobenzofuran]-2,19-dione(18a). Acid 17a (7.84 g, 12.9 mmol) was dissolved in 100 ml of anhydrousCH₂Cl₂ in the presence of 10 ml of triethylamine. PFP-TFA (9 ml, 52mmol) was added with stirring and the reaction was allowed to proceedfor 30 min. TLC analysis (9:1, CH₂Cl₂:MeOH) showed one major productwith R_(f)˜0.8. The solvent was evaporated to give a tan oil.Trituration with cold methanol (100 ml) produced a suspension of thedesired lactone 18a. After being stirred for ˜5 min the solid wascollected by filtration and washed with cold methanol. Drying undervacuum afforded 6.8 g (88%) of 18a as an off-white solid. ¹H NMR(DMSO-d₆): δ 11.27 (s, 1H), 7.43 (s, 1H), 7.20 (s, 1H), 6.95 (s, 1H),2.92 (t, J=7.2 Hz, 2H), 2.82 (t, J=7.2 Hz, 2H).

8,12,22,23,24,25-Hexachloro-9-hydroxyspiro[3,4-dihydro-2H-pyrano[3,2-b]xanthene-6,3′-3-hydroisobenzofuran]-2,19-dione(18b). ¹H NMR (DMSO-d₆): δ 11.36 (s, 1H), 7.44 (s, 1H), 7.24 (s, 1H),6.97 (s, 1H), 2.95 (m, 2H), 2.82 (m, 2H).

9-Hydroxyspiro[3,4-dihydro-2H-pyrano[3,2-b]xanthene-6,3′-3-hydroisobenzofuran]-2,19-dione(18c). ¹H NMR (DMSO-d₆): δ 10.21 (br s, 1H), 8.00 (d, J=7.2 Hz, 1H),7.75 (m, 2H), 7.28 (d, J=7.2 Hz, 1H), 7.12 (s, 1H), 6.76 (s, 1H), 6.70(s, 1H), 6.57 (s, 2H), 2.81 (t, J=7.5 Hz, 2H), 2.75 (m, 2H).

8,12,23,24-Tetrachloro-9-hydroxy-10-methylspiro[3,4-dihydro-2H-pyrano[3,2-b]xanthene-6,3′-3-hydroisobenzofuran]-2,19-dione(18d). ¹H NMR (DMSO-d₆): δ 10.05 (s, 1H), 8.31 (s, 1H), 7.89 (s, 1H),6.94 (s, 1H), 6.89 (s, 1H), 2.94 (t, J=7.5 Hz, 2H), 2.82 (t, J=7.5 Hz,2H), 2.39 (s, 3H).

Methyl3-(4,5,6,7,17-pentachloro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19a). 1,3-Dihydroxynaphthalene (0.57 g, 3.56 mmol) and 14a (1.0 g, 1.93mmol) were suspended in 10 ml of methanesulfonic acid. The suspensionwas stirred at 70° C. under argon for 1 h and cooled. A mixture of iceand water (50 g) was added to precipitate the product. Ethyl acetate (50ml) was added to extract the precipitate. The product from organic layerwas extracted with sodium bicarbonate solution. The aqueous phase waswashed with ether to remove excess 4-chlororesorcinol and acidified withconcentrated hydrochloric acid. The precipitated material, pure product16a, was taken up in ethyl acetate and dried over Na₂SO₄. Concentrationof the solution and drying under vacuum afforded 1.01 g (82%) of thetitle compound 19a as an orange solid. ¹H NMR (DMSO-d₆): 611.35 (s, 1H),10.20 (s, 1H), 8.23 (m, 1H), 7.40 (m, 2H), 6.96 (m, 1H), 6.87 (s, 1H),6.64 (s, 1H), 3.46 (s, 3H), 2.77 (t, J=7.2 Hz, 2H), 2.47 (t, J=7.2 Hz,2H).

Esters 19b-19i were prepared by the procedure described for compound19a.

Methyl3-(4,5,6,7,17-pentafluoro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19b). ¹H NMR (DMSO-d₆): 611.8 (br s, 1H), 10.8 (br s, 1H), 8.24 (m,1H), 7.46 (m, 2H), 7.12 (m, 1H), 6.80 (s, 1H), 6.63 (s, 1H), 3.52 (s,3H), 2.77 (t, J=7.2 Hz, 2H), 2.47 (t, J=7.2 Hz, 2H).

Methyl3-(4,5,6,7-tetrachloro-17-fluoro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19c). ¹H NMR (DMSO-d₆): 611.4 (br s, 1H), 10.6 (br s, 1H), 8.22 (m,1H), 7.40 (m, 2H), 6.96 (m, 1H), 6.89 (s, 1H), 6.48 (s, 1H), 3.46 (s,3H), 2.74 (t, J=7.2 Hz, 2H), 2.44 (t, J=7.2 Hz, 2H).

Methyl3-(17-chloro-4,5,6,7-tetrafluoro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19d). ¹H NMR (DMSO-d₆): δ 12.00 (bs, 1H), 11.39 (bs, 1H), 10.24 (bs,1H), 8.25 (m, 1H), 7.46 (bm, 2H), 7.10 (bm, 1H), 6.85 (bm, 1H), 6.78(bs, 1H), 3.52 (s, 3H), 2.80 (t, J=7.6 Hz, 2H), 2.49 (t, J=7.6 Hz, 2H).

Methyl3-(5,6,17-trichloro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19e). ¹H NMR (DMSO-d₆): δ 8.46 (s, 1H), 8.23 (d, J=8.5 Hz, 1H), 7.77(s, 1H), 7.31-7.42 (m, 2H), 6.90 (d, J=9.0 Hz, 1H), 6.78 (bs, 1H), 6.47(s, 1H), 3.45 (s, 3H), 2.73 (t, J=7.2 Hz, 2H), 2.44 (t, J=7.5 Hz, 2H).

Methyl3-(5,6-dichloro-17-fluoro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19f). ¹H NMR (DMSO-d₆): δ 11.8 (br s, 1H), 10.7 (br s, 1H), 8.46 (s,1H), 8.23 (d, J=8.5 Hz, 1H), 7.75 (s, 1H), 7.31-7.42 (m, 2H), 6.90 (d,J=9.0 Hz, 1H), 6.81 (bs, 1H), 6.30 (s, 1H), 3.45 (s, 3H), 2.70 (t, J=7.2Hz, 2H), 2.44 (t, J=7.5 Hz, 2H).

Methyl3-(4,5,6,7-tetrafluoro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19g). ¹H NMR (DMSO-d₆): δ 11.3 (br s, 1H), 10.5 (br s, 1H), 8.22 (d,J=8.5 Hz, 1H), 7.44 (m, 2H), 7.08 (m, 1H), 6.9-6.7 (m, 3H), 3.49 (s,3H), 2.72 (m, 2H), 2.49 (m, 2H).

Methyl3-(17-chloro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19h). ¹H NMR (DMSO-d₆): δ 11.3 (br s, 1H), 10.1 (br s, 1H), 8.19 (m,2H), 7.70 (m, 3H), 7.30 (m, 1H), 7.25 (m, 2H), 6.85 (m, 2H), 6.30 (s,1H), 3.40 (s, 3H), 2.70 (m, 2H), 2.38 (m, 2H).

Methyl3-(17-fluoro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoate(19i). ¹H NMR (DMSO-d₆): δ 11.23 (s, 1H), 10.44 (s, 1H), 8.17 (m, 2H),7.71 (m, 3H), 7.30 (m, 1H), 7.21 (m, 2H), 6.86 (m, 2H), 6.15 (s, 1H),3.41 (s, 3H), 2.66 (m, 2H), 2.38 (m, 2H).

3-(4,5,6,7,17-Pentachloro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoicacid (20a). Ester 19a (1.32 g, 2.06 mmol) was dissolved in 13 ml of 1NNaOH using sonication. After being stirred at room temperature for 1 h,the solution was acidified with 1N HCl to pH 3. The resultantprecipitate was collected by filtration and washed with water. Dryingunder vacuum over P₂O₅ afforded 1.1 g (85%) of acid 20a as an orangesolid. ¹H NMR (DMSO-d₆): δ 12.1 (br s, 1H), 11.34 (br s, 1H), 10.15 (brs, 1H), 8.22 (m, 1H), 7.40 (m, 2H), 6.95 (m, 1H), 6.87 (s, 1H), 6.74 (s,1H), 2.74 (m, 2H), 2.38 (m, 2H).

3-(17-Chloro-14,18-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,12′-benzo[a]xanthene]-19-yl)propanoicacid (20b). Acid 20b was prepared analogously to compound 20a. ¹H NMR(DMSO-d₆): δ 12.1 (br s, 1H), 11.23 (s, 1H), 10.04 (s, 1H), 8.18 (m,2H), 7.08 (m, 2H), 7.29 (m, 1H), 7.21 (m, 2H), 6.82 (m, 2H), 6.37 (s,1H), 2.66 (m, 2H), 2.29 (m, 2H).

4,5,6,7,17-Pentachloro-14-hydroxyspiro[3-hydroisobenzofuran-3,14′-benzo[f]chromano[7,6-b]4H-chromene]-1,19-dione(21a). Lactone 21a was prepared by the procedure described for compound18a. ¹H NMR (DMSO-d₆): δ 11.47 (br s, 1H), 8.24 (m, 1H), 7.42 (m, 2H),7.08 (s, 1H), 6.95 (m, 1H), 9.92 (s, 1H), 2.99 (m, 2H), 2.83 (m, 2H).

17-Chloro-14-hydroxyspiro[3-hydroisobenzofuran-3,14′-benzo[f]chromano[7,6-b]4H-chromene]-1,19-dione(21b). Lactone 21b was prepared by the procedure described for compound18a. ¹H NMR (DMSO-d₆): δ 11.39 (br s, 1H), 8.19 (m, 2H), 7.74 (m, 2H),7.4-7.2 (m, 3H), 6.95 (s, 1H), 6.55 (d, J=8.5 Hz, 1H), 6.66 (s, 1H),3.05 (m, 2H), 2.79 (m, 2H).

3-(4,5,6,7,20-Pentachloro-12,19-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,7′-benzo[h]xanthene]-18-yl)propanoicacid (22). Compound 22 was prepared from 18a and 1,6-dihydroxynaphtalenein 75% yield using the general method described for 19a. ¹H NMR(DMSO-d₆): δ 12.05 (br s, 1H), 10.24 (br s, 1H), 8.39 (d, 9 Hz, 1H),7.46 (d, J=9 Hz, 1H), 7.31 (d, J=9 Hz, 1H), 7.21 (s, 1H), 7.03 (s, 1H),6.99 (d, J=9 Hz, 1H), 2.74 (m, 2H), 2.40 (t, J=7.5 Hz, 2H).

4,5,6,7,22-Pentachloro-12-hydroxyspiro[3-hydroisobenzofuran-3,7′-benzo[h]chromano[7,6-b]4H-chromene]-1,20-dione(23). ¹H NMR (DMSO-d₆): δ 10.3 (br s, 1H), 8.39 (d, 9 Hz, 1H), 7.49 (d,J=9 Hz, 1H), 7.33 (m, 2H), 7.22 (s, 1H), 7.05 (d, J=9 Hz, 1H), 3.05 (m,2H), 2.79 (m, 2H).

Derivatization of Amino-Tailed Oligonucleotides Using Chromanone DyeDerivatives.

To a solution of triethylammonium salt of 5′-aminohexyl-octathymidylate(0.2 μmol) in 30 μL of DMSO was added 1 μmol of one of the lactone 8,12, 18 or 21 followed by 0.5 μL of triethylamine. The mixture wassonicated for a few seconds to dissolve the reagents, and the reactionwas allowed to proceed for 3-6 h. Progress of the reaction was monitoredby reverse-phase HPLC, an aliquot of 0.5 μL was typically taken for theanalysis. To precipitate the product (in some cases together with thederivatizing agent) 1 mL of 2% solution of LiClO₄ was added, theprecipitate was collected by centrifugation, washed with acetone anddried. The crude products were purified by denaturinggel-electrophoresis followed by reverse-phase HPLC.

5S,3R)-5-{[Bis(4-methoxyphenyl)phenylmethoxy]methyl}pyrrolidin-3-ol(25). To a stirred solution of Fmoc-DMT-prolinol (24) (Normand, H et alTetrahedron Lett., 1994, v.35; 51; 9509-9512.) (31.39 g, 48.9 mmol) inanhydrous CH₂Cl₂ (250 ml) was added 11.5 ml (76.7 mmol) of DBU. Afterbeing stirred at temperature for 0.5 h reaction mixture was concentratedand chromatographed on silica eluting first with Et₃N-MeOH—CH₂Cl₂(2:3:95) and then with Et₃N-MeOH—CH₂Cl₂ (2:10:88) mixture. Concentrationof the pure product fraction and drying under vacuum afforded 15.07 g(73%) of amine 25 as white foam.

Mixture of8-[3-((5S,3R)-5-([bis(4-methoxyphenyl)phenylmethoxy]methyl}-3-hydroxypyrrolidinyl)-3-oxopropyl]-7-oxophenoxazin-3-yl2,2-dimethylpropanoate and2-[3-((5S,3R)-5-{[bis(4-methoxyphenyl)phenylmethoxy]methyl}-3-hydroxypyrrolidinyl)-3-oxopropyl]-7-oxophenoxazin-3-yl2,2-dimethylpropanoate (27). Lactone 12a (7.5 g, 28.07 mmol) was addedin one portion to a solution of amine 25 (14.13 g, 433.68 mmol) in 150ml of anhydrous DMF. After being stirred at room temperature for 0.5 hdiisopropylethylamine (10.88 g, 14.67 ml, 84.2 mmol) was added followedby trimethylacetic anhydride (12.55 g, 16.37 ml, 67.36 mmol) and thereaction was stirred was stirred for another 1 h. The material obtainedafter evaporation of the solvent was chromatographed on silica elutingfirst with EtOAc:Et₃N (98:2) and then with a 0 to 5% gradient of MeOH inEtOAc. The fractions containing pure product were collected andconcentrated to give 21.7 g of an amorphous solid. To remove residualmethanol the solid was dissolved in EtOAc (60 ml) and precipitated byslowly adding to 600 ml of stirred hexane. The precipitate was filteredoff and washed with hexane. Drying under vacuum afforded 17.9 g (83%) of27 as a yellow-orange solid (9:1 mixture of isomers).

A mixture of8-[3-((2S,4R)-2-{[1,1-bis(4-methoxyphenyl)-2-ethylidenebut-3-enyloxy]methyl}-4-{(2-cyanoethoxy)[ethyl(methylethyl)amino]phosphinooxy}-pyrrolidinyl)-3-oxopropyl]-7-oxophenoxazin-3-yl2,2-dimethylpropanoate and2-[3-((2S,4R)-2-{[bis(4-methoxyphenyl)phenylmethoxy]methyl}-4-{[bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy}pyrrolidinyl)-3-oxopropyl]-7-oxophenoxazin-3-yl2,2-dimethylpropanoate (28). To a solution of 27 (10.5 g, 13.62 mmol) inanhydrous CH₂Cl₂ (200 ml) was added with stirring diisopropylethylamine(6.99 ml, 5.187 g, 40.13 mmol) followed by2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (4.22 g, 3.98 ml,17.84 mmol). After being stirred at room temperature for 2 h, methanol(0.2 ml, 4.9 mmol) was added to quench excess chlorophosphoramidite. Thereaction mixture was concentrated and loaded on a silica gel columnwhich had been pre-washed with EtOAc:Hexane:Et₃N (60:40:5) mixture (1 L)and then equilibrated with EtOAc:Hexane (3:2) mixture. The product waseluted a 10-60% gradient of ethyl acetate in hexane. Concentration ofthe pure product fractions gave the desired phosphoramidite as an orangefoam. To obtain product as a solid, the foam was dissolved in anhydrousether (20 ml) and precipitated by dropwise addition to vigorouslystirred hexane (700 ml) under argon. The precipitate was filtered offand dried in vacuo to afford (8.9 g) of 28 (67%) as an amorphous solid.³¹P NMR (DMSO-d₆): δ 143.64, 143.04.

Methyl 3-(2,6-dihydroxyphenyl)propionate (30) was prepared by treatingmethanolic solution of 3-(2,6-dihydroxyphenyl)propionic acid (29)(Mitoshi K. et al. Syn. Lett., 12; 1997; 1472-1474) with anhydrous HClgas. ¹H NMR (d6-DMSO) δ: 9.16 (s, 2H), 6.76 (t, J=8 Hz, 1H), 6.26 (d,J=8 Hz, 2H), 3.59 (s, 3H), 2.75 (t, J=7.5 Hz, 2H), 2.34 (t, J=7.5 Hz,2H).

Methyl 3-(3-chloro-2,6-dihydroxyphenyl)propionate (31). To a solution of30 (5.0 g, 25.5 mmol) in 50 ml of methanol was added 3.4 g (25.5 mmol)of N-chlorosuccinimide. The reaction was stirred overnight at roomtemperature and concentrated. The residue was suspended in a mixture ofhexane and ethyl acetate and the precipitated succinimide was removed byfiltration. Crude product obtained after evaporation of the solvent waschromatographed on silica eluting with 4:1 hexane-ethyl acetate.Concentration of the pure product fractions afforded 4.4 g of the titlecompound as colorless oil that slowly crystallized during prolongeddrying under vacuum. ¹H NMR (d6-DMSO) δ: 9.52 (s, 1H), 8.95 (s, 1H),6.97 (d, J=8.5 Hz, 1H), 6.35 (d, J=8.5 Hz, 1H), 3.59 (s, 3H), 2.81 (t,J=7.5 Hz, 2H), 2.34 (t, J=7.5 Hz, 2H).

4,5-Dichloro-2-({5-chloro-2,4-dihydroxy-3-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoicacid (35). Benzophenone 35 was synthesized according to the generalprocedure described for compound 14a in a similar synthetic yield. ¹HNMR (d6-DMSO) δ: 12.25 (s, 1H), 10.69 (br s, 1H), 8.14 (s, 1H), 7.90 (s,1H), 7.06 (s, 1H), 3.61 (s, 3H), 2.93 (t, J=7.5 Hz, 2H), 2.48 (t, J=7.5Hz, 2H).

3-(5,6,11,16-Tetrachloro-12,15-dihydroxy-14-methyl-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-13-yl)propanoicacid (37). To a suspension of 35 (200 mg, 0.45 mmol) and2-methyl-4-chlororesorcinol (110 mg, 0.69 mmol) in 5 ml oftrifluoroacetic acid was added 0.7 ml of methanesulfonic acid. Thereaction was stirred at room temperature for 6 h to give a dark-purplesolution. The solvent was removed on a rotary evaporator at roomtemperature and water was added to precipitate the colorlessintermediate 36. The solid was collected by filtration, washed withwater and re-suspended in 15 ml of water. Sodium hydroxide solution (5ml, 1N) was added and the resultant solution was kept at roomtemperature for 20 min and acidified with 1N HCl to pH of 2. Theprecipitated product was filtered off and washed with water. Dryingunder vacuum afforded 202 mg (81%) of the desired dye as an orangesolid. ¹H NMR (d6-DMSO) δ: 12.25 (s, 1H), 9.96 (br s, 1H), 8.14 (s, 1H),7.82 (s, 1H), 7.05 (s, 1H), 6.82 (s, 1H), 3.14 (m, 2H), 2.87 (m, 2H),2.35 (s, 3H).

5,9,23,24-Tetrachloro-10-hydroxy-11-methylspiro[1,2-dihydro-2H-pyrano[2,3-c]xanthene-7,3′-3-hydroisobenzofuran]-3,19-dione(38). Lactone 38 was synthesized according to the general methoddescribed for compound 18a. ¹H NMR (d6-DMSO) δ: 10.03 (s, 1H), 8.27 (s,1H), 7.85 (s, 1H), 7.13 (s, 1H), 6.85 (s, 1H), 3.28 (m, 2H), 2.39 (m,2H), 2.36 (s, 3H)

11-[2-(N-{6-[Bis(4-methoxyphenyl)phenylmethoxy]hexyl}carbamoyl)ethyl]-15-(2,2-dimethylpropanoyloxy)-5,6,13,16-tetrachloro-14-methyl-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-12-yl2,2-dimethylpropanoate (41). A solution of 18d (2.67 g, 4.9 mmol),5-aminohexanol (0.7 g, 5.4 mmol) and triethylamine (5 ml) in 20 ml ofanhydrous DMF was kept at room temperature for 5 h. TLC analysis (1:9,MeOH:CH₂Cl₂) showed complete conversion of 18d into a new, lowermobility product (compound 39). DMTr-chloride (6.0 g) was added inseveral portions over several hours until all the intermediate 39 wasconverted into compound 40 (analysis by TLC).

N-Methylimidazole (5 ml) and trimethylacetic anhydride (6 ml) wereadded. The reaction was heated at 50° C. with stirring for 7 h. Thesolvent was evaporated under vacuum and the resultant oil waspartitioned between water (200 ml) and ethyl acetate (150 ml). Theorganic phase was washed with brine and dried over Na₂SO₄. The materialobtained after solvent concentration was chromatographed on silicaeluting with 3:1 hexane-ethyl acetate. Concentration of the productcontaining fractions afforded 3.4 g (61%) of 41 as a pale yellow,amorphous solid. ¹H NMR (DMSO-d₆): δ 8.32 (s, 1H), 8.0 (br s, 1H), 7.66(t, J=5 Hz, 1H), 7.4-7.1 (m, 10H), 6.90)s, 1H), 6.88 (s, 2H), 6.87 (s,2H), 3.72 (s, 6H), 2.9 (m, 4H), 2.58 (m, 2H), 2.30 (s, 3H), 1.49 (m),1.38 (s, 18H), 1.27 (m), 1.16 (m).

15-(2,2-Dimethylpropanoyloxy)-5,6,13,16-tetrachloro-11-{2-[N-(6-hydroxyhexyl)carbamoyl]ethyl}-14-methyl-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-12-yl2,2-dimethylpropanoate (42). To a solution of 41 (3.4 g, 3 mmol) in 50ml of 10% methanol in dichloromethane was added 0.15 ml oftrifluoroacetic acid. After being stirred for 1 h, the solution wastreated with triethylamine (0.15 ml) to neutralize the acid. Thereaction mixture was chromatographed on a silica gel column eluting withethyl acetate. Concentration of the pure product fraction afforded 2.0 g(73%) of the title compound (42) as an off-white, amorphous solid. ¹HNMR (DMSO-d₆): δ 7.75 (s, 1H), 7.73 (br t, 1H), 7.69 (s, 1H), 7.19 (s,1H), 3.34 (t, J=6.5 Hz, 2H), 2.89 (m, 2H), 2.59 (m, 2H), 2.20 (m, 2H),1.38 (s, 9H), 1.34 (s, 9H), 1.4-1.1 (m overlapping with two (CH₃)₃signals, 6H).

11-{2-[N-(6-{[Bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy}hexyl)carbamoyl]ethyl}-15-(2,2-dimethylpropanoyloxy)-5,6,13,16-tetrachloro-14-methyl-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-12-yl2,2-dimethylpropanoate (43). To a stirred solution of 42 (1.2 g, 1.45mmol) in 10 ml of anhydrous CH₂Cl₂ was added diisopropylethylamine (0.5ml) followed by 0.35 ml (1.5 mmol) of 2-cyanoethyl-N,N-diisipropylchlorophosphoramidite. After being stirred for 1 h, the reaction wasquenched with methanol (0.05 ml) and diluted with ethyl acetate (50 ml).The solution was washed with saturated sodium bicarbonate, brine anddried over Na₂SO₄. The crude material obtained after evaporation of thesolvent was purified by precipitation in anhydrous hexane from smallamount of ether. The precipitated solid was collected by filtration andwashed with hexane. Drying under vacuum afforded 1.1 g (74%) of 43 as acolorless, amorphous solid. ¹H NMR (DMSO-d₆): δ

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A method for preparing a fluorescent dye-labeled biological agent,said method comprising contacting an unlabeled biological agent with afluorescent dye-fused lactone derivative under conditions sufficient tocovalently attach the fluorescent dye to said biological agent and forma fluorescent dye-labeled biological agent.
 2. A method for preparing afluorescent dye-labeled phosphoramidite reagent, said method comprising:(a) contacting a fluorescent dye-fused lactone derivative with a linkinggroup component to form an intermediate fluorescent dye-labeled linkinggroup; and (b) contacting said intermediate fluorescent dye-labeledlinking group with a phosphoramidite moiety under conditions sufficientto covalently attach the phosphoramidite moiety to said fluorescentdye-labeled linking group and form said fluorescent dye-labeledphosphoramidite reagent.
 3. A method in accordance with claim 2, whereinsaid fluorescent dye-fused lactone derivative has a formula selectedfrom the group consisting of:

wherein A¹ represents O or N—Z in which Z is H or (C₁-C₈)alkyl, or isoptionally combined with R² or R¹ to form a 5- or 6-membered ring or iscombined with each of R² and R⁴ to form two fused 6-membered rings; oris optionally combined with and adjacent X⁰ to form a 5- or 6-memberedring or is combined with two adjacent X⁰ to form two fused 6-memberedrings; R^(1′), R¹, R², R³ and R⁴ are each independently selected from H,halogen, cyano, CF₃, C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryland heteroaryl; R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently selectedfrom H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of X⁰, R^(1′)and R¹ through R⁹ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl portions of any of X⁰, R^(1′) and R¹ throughR⁹ are optionally substituted with from one to four substituentsselected from the group consisting of halogen, cyano, carboxy, sulfo,hydroxy, amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl,(C₁-C₆)alkylthio and (C₁-C₆)alkoxy; the subscript n is an integer offrom 0 to 4; each X⁰ is a member independently selected from the groupconsisting of H, halogen, cyano, CF₃, (C₁₋₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl, heteroaryl, SO₃Hand CO₂H; R⁰ is a member selected from the group consisting of halogen,cyano, CF₃, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, substituted orunsubstituted heteroaryl or aryl having the formula:

wherein X¹, X², X³, X⁴ and X⁵ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, SO₃H and CO₂H, andoptionally, any two adjacent X¹ through X⁵ are combined to form anaromatic or heteroaromatic ring.
 4. A method in accordance with claim 2,wherein fluorescent dye-fused lactone derivative has a formula selectedfrom the group consisting of:

wherein A is hydroxy, amino, protected hydroxy, or protected amino; thesubscript n is an integer of from 0 to 4; each X⁰ is a memberindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, aryl, heteroaryl, SO₃H and CO₂H; R^(1′), R¹, R², R³ andR⁴ are each independently selected from H, halogen, cyano,CF_C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy; R⁵, R⁶, R⁷ and R⁸are each independently selected from H, (C₁-C₈)alkyl, aryl andaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of X⁰, R^(1′) and R¹through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl portions of any of X⁰, R^(1′) and R¹ through R⁸ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁₋₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; and optionally, any two adjacent substituents X¹ throughX⁴ can be taken together to form a fused aromatic or heteroaromatic ringthat is optionally further substituted with from one to foursubstituents selected from halogen cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C_-C₆)alkoxy.
 5. A method in accordance with claim 2, wherein saidlinking group component comprises two reactive functional groupsselected from amino, hydroxy, hydrazino and thiol.
 6. A method inaccordance with claim 2, wherein said linking group is linear or cyclicor a combination thereof.
 7. A method in accordance with claim 2,wherein said linking group comprises a (C₂-C₂₀)alkylene or(C₂-C₂₀)heteroalkylene group.
 8. A method in accordance with claim 2,wherein said linking group is cyclic and comprises a five-memberedheterocycle.
 9. A method in accordance with claim 8, wherein said cycliclinking group is a prolinol linker.
 10. A method in accordance withclaim 1, wherein said biological agent is an oligonucleotide.
 11. Amethod in accordance with claim 1, wherein said biological agent is anoligonucleotide having one or more modified bases.
 12. A method inaccordance with claim 1, wherein said fluorescent dye-fused lactonederivative is a member selected from the group consisting of coumarins,benzocoumarins, xanthenes, benzo[a]xanthenes, benzo[b]xanthenes,benzo[c]xanthenes, phenoxazines, benzo[a]phenoxazines,benzo[b]phenoxazines and benzo[c]phenoxazines.
 13. A method inaccordance with claim 1, wherein said fluorescent dye-fused lactonederivative has a formula selected from the group consisting of:

wherein R¹ and R^(1′) are each members independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; R⁵, R⁶, R⁷ and R⁸are each independently selected from the group consisting of H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹, R^(1′),and R⁵ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of R¹, R^(1′),and R⁵ through R⁸ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; R^(A) and R^(B) arecombined to form a substituted or unsubstituted fused ring system havingfrom 1 to 4 five- or six-membered rings; with the proviso that thecompound has an emission wavelength of from 400 nm to 1200 nm.
 14. Amethod in accordance with claim 13, wherein said fluorescent dye-fusedlactone derivative has the formula:

wherein A¹ represents O or N—Z in which Z is H or (C₁-C₈)alkyl, or isoptionally combined with R² or R⁴ to form a 5- or 6-membered ring or iscombined with each of R² and R⁴ to form two fused 6-membered rings;R^(1′), R¹, R², R³ and R⁴ are each independently selected from H,halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryland heteroaryl; R⁵, R⁶, R⁷ and R⁸ are each independently selected fromH, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′) andR¹ through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl portions of any of R^(1′) and R¹ through R⁸ are optionallysubstituted with from one to four substituents selected from the groupconsisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;R⁰ is halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl,substituted or unsubstituted heteroaryl or aryl having the formula:

wherein X¹, X², X³, X⁴ and X⁵ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, SO₃H and CO₂H, andoptionally, any two adjacent X¹ through X⁵ are combined to form anaromatic or heteroaromatic ring.
 15. A method in accordance with claim14, wherein R¹ is selected from the group consisting of H, halogen andphenyl; and each R², R³ and R⁴ is independently selected from the groupconsisting of H and halogen.
 16. A method in accordance with claim 14,wherein said fluorescent dye-fused lactone derivative has a formulaselected from the group consisting of:

wherein A is hydroxy, amino, protected hydroxy, or protected amino;R^(1′), R¹, R², R³ and R⁴ are each independently selected from H,halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy;R⁵, R⁶, R⁷ and R⁸ are each independently selected from H, (C₁-C₈)alkyl,aryl and aryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′)and R¹ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl portions of any of R⁵ through R⁸ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; and optionally, any two adjacent substituents X¹ throughX⁴ can be taken together to form a fused aromatic or heteroaromatic ringthat is optionally further substituted with from one to foursubstituents selected from halogen cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy.
 17. A method in accordance with claim 16, wherein saidfluorescent dye-fused lactone derivative has formula Ia in which A ishydroxy or a protected hydroxy; R⁵ through R⁸ are each H; R¹, R², R³, R⁴and R^(1′) are independently selected from the group consisting of H,halogen, cyano and CF₃.
 18. A method in accordance with claim 13,wherein said fluorescent dye-fused lactone derivative has the formula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl, or is optionallycombined with R⁴ to form a 5- or 6-membered ring; R^(1′), R¹ and R⁴ areeach independently selected from H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; R⁵, R⁶, R⁷ and R⁸are each independently selected from H, (C₁-C₈)alkyl, aryl, heteroaryl,aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; wherein the alkyl portionsof any of R^(1′) and R¹ through R⁸ are optionally substituted withhalogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano,haloacetyl or hydroxy, and the alkyl portions of the substituents havefrom 1 to 6 carbon atoms; and the aryl portions of any of R^(1′) and R¹through R⁸ are optionally substituted with from one to four substituentsselected from the group consisting of halogen, cyano, carboxy, sulfo,hydroxy, amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl,(C₁-C₆)alkylthio and (C₁-C₆)alkoxy; the subscript n is an integer offrom 0 to 4; each X⁰ is a member independently selected from the groupconsisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl, heteroaryl, SO₃Hand CO₂H; R⁰ is halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkenyl,(C₁-C₈)alkynyl, substituted or unsubstituted heteroaryl or aryl havingthe formula:

wherein X¹, X², X³, X⁴ and X⁵ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, SO₃H and CO₂H, andoptionally, any two adjacent X¹ through X⁵ are combined to form anaromatic or heteroaromatic ring.
 19. A method in accordance with claim18, wherein said has a formula selected from the group consisting of:

wherein A is hydroxy, amino, protected hydroxy, or protected amino;R^(1′), R¹and R⁴ are each independently selected from H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy; R⁵, R⁶, R⁷ and R⁸are each independently selected from H, (C₁-C₈)alkyl, aryl andaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′) and R¹through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl portions of any of R⁵ through R⁸ are optionally substitutedwith from one to four substituents selected from the group consisting ofhalogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;and optionally, any two adjacent substituents X¹ through X⁴ can be takentogether to form a fused aromatic or heteroaromatic ring that isoptionally further substituted with from one to four substituentsselected from halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy.20. A method in accordance with claim 19, wherein said fluorescentdye-fused lactone derivative has formula IIa in which A is hydroxy or aprotected hydroxy; R⁵ through R⁸ are each H; R¹, R⁴ and R^(1′) areindependently selected from the group consisting of H, halogen, cyanoand CF₃.
 21. A method in accordance with claim 12, wherein saidbenzo[b]xanthene has the formula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl, or is optionallycombined with an adjacent X⁰ to form a 5- or 6-membered ring or iscombined with two adjacent X) groups to form two fused 6-membered rings;R^(1′), R¹, R³ and R⁴ are each independently selected from H, halogen,cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl andheteroaryl; R⁵, R⁶, R⁷ and R⁸ are each independently selected from H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′) andR¹ through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl portions of any of R^(1′) and R¹ through R⁸ are optionallysubstituted with from one to four substituents selected from the groupconsisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;the subscript n is an integer of from 0 to 4; each X⁰ is a memberindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl,(C₁-C₈)alkynyl, aryl, heteroaryl, SO₃H and CO₂H; R⁰ is a member selectedfrom the group consisting of halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkenyl, (C₁-C₈)alkynyl, and substituted or unsubstitutedheteroaryl or aryl having the formula:

wherein X¹, X², X³, X⁴ and X⁵ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, SO₃H and CO₂H, andoptionally, any two adjacent X¹ through X⁵ are combined to form anaromatic or heteroaromatic ring.
 22. A method in accordance with claim21, wherein R¹ is halogen; and each X¹ through X⁴ is independentlyselected from the group consisting of H, F and Cl.
 23. A method inaccordance with claim 21, wherein said benzo[b]xanthene has a formulaselected from the group consisting of:

wherein A is hydroxy, amino, protected hydroxy, or protected amino; thesubscript n is an integer of from 0 to 3; each X⁰ is a memberindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl,(C₁-C₈)alkynyl, aryl, heteroaryl, SO₃H and CO₂H; R^(1′), R¹, R³ and R⁴are each independently selected from H, halogen, cyano, CF₃,(C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy; R⁵, R⁶, R⁷ and R⁸ areeach independently selected from H, (C₁-C₈)alkyl, aryl andaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′) and R¹through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl portions of any of R⁵ through R⁸ are optionally substitutedwith from one to four substituents selected from the group consisting ofhalogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;and optionally, any two adjacent substituents X¹ through X⁴ can be takentogether to form a fused aromatic or heteroaromatic ring that isoptionally further substituted with from one to four substituentsselected from halogen cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy.24. A method in accordance with claim 23, wherein said fluorescentdye-fused lactone derivative has formula IIIa in which A is hydroxy or aprotected hydroxy; R⁵ through R⁸ are each H; R¹, R³, R⁴ and R^(1′) areindependently selected from the group consisting of H, halogen, cyanoand CF₃.
 25. A method in accordance with claim 12, wherein saidbenzo[c]xanthene has the formula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl, or is optionallycombined with an adjacent X⁰ to form a 5- or 6-membered ring or iscombined with two adjacent X⁰ groups to form two fused 6-membered rings;R^(1′), R¹and R⁴ are each independently selected from H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl;R⁵, R⁶, R⁷ and R⁸ are each independently selected from H, (C₁-C₈)alkyl,aryl, heteroaryl, aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; whereinthe alkyl portions of any of R^(1′) and R¹ through R⁸ are optionallysubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms; and the aryl portions of anyof R^(1′) and R¹ through R⁸ are optionally substituted with from one tofour substituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; the subscript n is aninteger of from 0 to 3; each X⁰ is a member independently selected fromthe group consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl,heteroaryl, SO₃H and CO₂H; R⁰ is halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkenyl, (C₁-C₈)alkynyl, substituted or unsubstituted heteroarylor aryl having the formula:

wherein X¹, X², X³, X⁴ and X⁵ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C8)alkenyl, (C₁-C₈)alkynyl, SO₃H and CO₂H, andoptionally, any two adjacent X through X⁵ are combined to form anaromatic or heteroaromatic ring.
 26. A method in accordance with claim25, wherein said benzo[c]xanthene has a formula selected from the groupconsisting of:

wherein A is hydroxy, amino, protected hydroxy, or protected amino; thesubscript n is an integer of from 0 to 3; each X⁰ is a memberindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl,(C₁-C₈)alkynyl, aryl, heteroaryl, SO₃H and CO₂H; R^(1′), R¹, R³ and R⁴are each independently selected from H, halogen, cyano, CF₃,(C₁-C₈)alkyl, (C₁-C₈)alkylthio and (C₁-C₈)alkoxy; R⁵, R⁶, R⁷ and R⁸ areeach independently selected from H, (C₁-C₈)alkyl, aryl andaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′) and R¹through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl portions of any of R⁵ through R⁸ are optionally substitutedwith from one to four substituents selected from the group consisting ofhalogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;and optionally, any two adjacent substituents X¹ through X⁴ can be takentogether to form a fused aromatic or heteroaromatic ring that isoptionally further substituted with from one to four substituentsselected from halogen cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy.27. A method in accordance with claim 26, wherein said fluorescentdye-fused lactone derivative has formula IIa in which A is hydroxy or aprotected hydroxy; R⁵ through R⁸ are each H; R¹, R², R³ and R^(1′) areindependently selected from the group consisting of H, halogen, cyanoand CF₃.
 28. A method in accordance with claim 12, wherein saidphenoxazine has the formula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl, or is optionallycombined with R² or R⁴ to form a 5- or 6-membered ring or is combinedwith each of R² and R⁴ to form two fused 6-membered rings; R¹, R², R³,R⁴ and R⁹ are each independently selected from the group consisting ofH, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy,aryl and heteroaryl; R⁵, R⁶, R⁷ and R⁸ are each independently selectedfrom H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹ throughR⁹ are optionally substituted with halogen, carboxy, sulfo, amino, mono-or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and the arylportions of any of R¹ through R⁹ are optionally substituted with fromone to four substituents selected from the group consisting of halogen,cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy.
 29. A method inaccordance with claim 28, wherein said fluorescent dye-fused lactonederivative has formula V in which A¹ is O; R⁵ through R⁸ are each H; R¹through R⁴ and R⁹ are independently selected from the group consistingof H, halogen, cyano and CF₃.
 30. A method in accordance with claim 12,wherein said benzo[a]phenoxazines have the formula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl, or is optionallycombined with R⁴ to form a 5- or 6-membered ring; R¹, R⁴ and R⁹ are eachindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl;R⁵, R⁶, R⁷ and R⁸ are each independently selected from the groupconsisting of H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹ throughR⁹ are optionally substituted with halogen, carboxy, sulfo, amino, mono-or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and the arylportions of any of R¹ through R⁹ are optionally substituted with fromone to four substituents selected from the group consisting of halogen,cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; the subscript n is aninteger of from 0 to 4; and each X⁰ is independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl, heteroaryl, SO₃Hand CO₂H.
 31. A method in accordance with claim 30, wherein saidfluorescent dye-fused lactone derivative has formula VI in which A¹ isO; R⁵ through R⁸ are each H; R¹, R⁴ and R⁹ are independently selectedfrom the group consisting of H, halogen, cyano and CF₃.
 32. A method inaccordance with claim 12, wherein said benzo[b]phenoxazines have theformula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl, or is optionallycombined with an adjacent X⁰ to form a 5- or 6-membered ring or iscombined with two adjacent X⁰ groups to form two fused 6-membered rings;R¹, R³, R⁴ and R⁹ are each independently selected from H, halogen,cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl andheteroaryl; R⁵, R⁶, R⁷ and R⁸ are each independently selected from thegroup consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyland heteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹ orR³ through R⁹ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl or heteroaryl portions of any of R¹ and R³ through R⁹ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; each X⁰ is independently selected from the groupconsisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, aryl, heteroaryl, SO₃Hand CO₂H. Additionally, the alkyl portions of any X⁰ can be furthersubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms; and the subscript n is aninteger of from 0 to
 3. 33. A method in accordance with claim 32,wherein said fluorescent dye-fused lactone derivative has formula VII inwhich A¹ is O; R⁵ through R⁸ are each H; R¹, R³, R⁴ and R⁹ areindependently selected from the group consisting of H, halogen, cyanoand CF₃.
 34. A method in accordance with claim 12, wherein saidbenzo[c]phenoxazines have the formula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl, or is optionallycombined with an adjacent X⁰ to form a 5- or 6-membered ring or iscombined with two adjacent X⁰ groups to form two fused 6-membered rings;R¹, R², R³ and R⁹ are each independently selected from H, halogen,cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl andheteroaryl; R⁵, R⁶, R⁷ and R⁸ are each independently selected from thegroup consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyland heteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹, R²,R³ or R⁵ through R⁹ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of R¹, R², R³or R⁵ through R⁹ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; each X⁰ isindependently selected from the group consisting of H, halogen, cyano,CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, (C₁-C₈)alkenyl,(C₁-C₈)alkynyl, aryl, heteroaryl, SO₃H and CO₂H, wherein the alkyl oraryl portions of any X⁰ can be further substituted with halogen,carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetylor hydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the subscript n is an integer of from 0 to
 3. 35. Amethod in accordance with claim 34, wherein said fluorescent dye-fusedlactone derivative has formula VIII in which A¹ is O; R⁵ through R⁸ areeach H; R¹, R², R³ and R⁹ are independently selected from the groupconsisting of H, halogen, cyano and CF₃.
 36. A method in accordance withclaim 13, wherein said substituted coumarin has the formula:

wherein R¹, R^(1′), R² and R³ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; R⁵, R⁶, R⁷ and R⁸are each independently selected from the group consisting of H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R^(1′) orR¹ through R⁸ are optionally substituted with halogen, carboxy, sulfo,amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, andthe alkyl portions of the substituents have from 1 to 6 carbon atoms;and the aryl or heteroaryl portions of any of R^(1′) and R¹ through R⁸are optionally substituted with from one to four substituents selectedfrom the group consisting of halogen, cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy, or optionally, R² and R³ are taken together to form afused aromatic ring; A¹ represents O or N—Z, in which Z is H or(C₁-C₈)alkyl.
 37. A method in accordance with claim 36, wherein R¹ is Hor halogen; R³ is (C₁-C₄)alkyl; and R^(1′) and R² are each hydrogen. 38.A method in accordance with claim 36, wherein R² and R³ areindependently selected from halogen, cyano, CF₃, (C₁-C₈)alkyl, and arylor heteroaryl having the formula:

wherein X¹, X², X³, X⁴ and X⁵ are each independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₁-C₈)alkenyl, (C₁-C₈)alkynyl, SO₃H and CO₂H, whereinthe alkyl portions of any of X¹ through X⁵ can be further substitutedwith halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy,cyano, haloacetyl or hydroxy, and the alkyl portions of the substituentshave from 1 to 6 carbon atoms, and optionally, any two adjacentsubstituents X¹ through X⁵ can be taken together to form a fusedaromatic ring that is optionally further substituted with from one tofour substituents selected from halogen cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy.
 39. A fused lactone dye having a formula selected from:

wherein R¹ and R^(1′) are each members independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; R⁵, R⁶, R⁷ and R⁸are each independently selected from the group consisting of H,(C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹, R^(1′),and R⁵ through R⁸ are optionally substituted with halogen, carboxy,sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl orhydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl or heteroaryl portions of any of R¹, R^(1′),and R⁵ through R⁸ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; R^(A) and R^(B) arecombined to form a substituted or unsubstituted fused ring system havingfrom 1 to 4 five- or six-membered rings; with the proviso that thecompound has an emission wavelength of from 400 nm to 1200 nm.
 40. Afused-lactone dye of claim 39, having a formula selected from the groupconsisting of formula I, II, III, IV, V, VI, VII, VIII and IX.
 41. Afused-lactone dye of claim 39, having a formula selected from the groupconsisting of formula Ia, Ib, Ia, IIb, IIIa, IIIb, IVa, IVb, V and VI.42. A dye reagent having a formula selected from:

wherein R¹ and R^(1″) are each members independently selected from thegroup consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; each R⁵ and R⁶ isindependently selected from the group consisting of H, (C₁-C₈)alkyl,aryl, heteroaryl, aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; whereinthe alkyl portions of any of R¹, R^(1′), R⁵ and R⁶ are optionallysubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms; and the aryl or heteroarylportions of any of R¹, R^(1′), R⁵ and R⁶ are optionally substituted withfrom one to four substituents selected from the group consisting ofhalogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;R^(A) and R^(B) are combined to form a substituted or unsubstitutedfused ring system having from 1 to 4 five- or six-membered rings; R^(x)is selected from the group consisting of H and hydroxy protectinggroups; the subscript p is an integer of from 1 to 3; W is a di-, tri-or tetravalent linker which is acyclic, cyclic, aromatic or acombination thereof, having from 4 to 50 atoms selected from the groupconsisting of C, N, O, P and S and exculsive of hydrogen atoms that fillavailable valences, and further having a nitrogen atom directlyconnected to the adjacent carbonyl group; K is selected from the groupconsisting of a lone pair of electrons, H, OH, SH, NH, (C₁-C₈)alkyl,aryl, an amino protecting group and and a hydroxy protecting group; thesubscript n is 0 or 1; and when n is 1, Y is a cleavable linking groupand L is a solid support; and when n is 0, L is a phosphoramidite orreactive functional group the subscript p is an integer of from 1 to 3;with the proviso that the reagent has an emission wavelength of from 400to 1200 nm.
 43. A dye reagent in accordance with claim 42, wherein n is1, and Y is selected from the group consisting of:

wherein the subscripts q and r are independently integers of from 1 to15; and each R is independently (C₁-C₈)alkyl or (C₁-C₈)alkoxy.
 44. A dyereagent in accordance with claim 42, wherein said reagent has anemission wavelength of from 400 nm to about 850 nm.
 45. A dye reagent inaccordance with claim 42, wherein R^(A), R^(B) and the ring to whicheach is attached forms a dye selected from the group consisting of afluorescein, a benzocoumarin, a xanthene, a benzo[a]xanthene, abenzo[b]xanthene, a benzo[c]xanthene, a phenoxazine, abenzo[a]phenoxazine, a benzo[b]phenoxazine and a benzo[c]phenoxazine.46. A dye reagent in accordance with claim 45, having the formula:

wherein A¹ is O or N—Z in which Z is H or (C₁-C₈)alkyl; R¹, R², R³, R⁴and R⁹ are each independently selected from the group consisting of H,halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryland heteroaryl; each R⁵ and R⁶ is independently selected from the groupconsisting of H, (C₁-C₈)alkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)alkyl; wherein the alkyl portions of any of R¹ throughR⁹ are optionally substituted with halogen, carboxy, sulfo, amino, mono-or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and the arylportions of any of R¹ through R⁹ are optionally substituted with fromone to four substituents selected from the group consisting of halogen,cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; optionally, R² takentogether with R³ form a fused aromatic ring that is optionallysubstituted with from one to four substituents selected from halogencyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; and tautomeric formsthereof.
 47. A dye reagent in accordance with claim 46, having theformula:

wherein R^(y) is a protecting group.
 48. A dye reagent of claim 47,having the formula:


49. A 5,6-dichlorofluorescein dye reagent having the formula:

wherein R^(x) and R^(y) are H or independently selected protectinggroups; R^(1′), R¹, R², R³ and R⁴ are each independently selected fromthe group consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; each R⁵ and R⁶ isindependently selected from the group consisting of H, (C₁-C₈)alkyl,aryl, heteroaryl, aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; whereinthe alkyl portions of any of R^(1′) and R¹ through R⁶ are optionallysubstituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino,alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of thesubstituents have from 1 to 6 carbon atoms; and the aryl portions of anyof R^(1′) and R¹ through R⁶ are optionally substituted with from one tofour substituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; optionally, R² takentogether with R³ form a fused aromatic or heteroaromatic ring that isoptionally substituted with from one to four substituents selected fromhalogen cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;the subscript p is an integer of from 1 to 3; W is a di-, tri- ortetra-valent linker which is acyclic, cyclic, aromatic or a combinationthereof, having from 4 to 50 atoms selected from the group consisting ofC, N, O, P and S and exculsive of hydrogen atoms that fill availablevalences, and further having a nitrogen atom directly connected to theadjacent carbonyl group; K is selected from the group consisting of alone pair of electrons, H, OH, SH, NH, (C₁-C₈)alkyl, aryl, an aminoprotecting group and and a hydroxy protecting group; the subscript n is0 or 1; and when n is 1, Y is a cleavable linking group and L is a solidsupport; and when n is 0, L is a phosphoramidite or reactive functionalgroup the subscript p is an integer of from 1 to 3; and tautomeric formsthereof.
 50. A 5,6-dichlorofluorescein dye reagent in accordance withclaim 49, wherein R¹ is Cl; R⁵ and R⁶ are H; and the subscript p is 2.51. A 5,6-dichlorofluorescein dye reagent in accordance with claim 50,having a formula selected from:


52. A leuco-phenoxazin e dye reagent having the formula:

wherein each of R_(x), R_(y) and R_(z) are protecting groups; R¹, R²,R³, R⁴ and R⁹ are each independently selected from the group consistingof H, halogen, cyano, CF₃, (C₁-C₈)alkyl, (C₁-C₈)alkylthio,(C₁-C₈)alkoxy, aryl and heteroaryl; each R⁵ and R⁶ is independentlyselected from the group consisting of H, (C₁-C₈)alkyl, aryl, heteroaryl,aryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)alkyl; wherein the alkyl portionsof any of R¹ through R⁹ are optionally substituted with halogen,carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetylor hydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and the aryl portions of any of R¹ through R⁹ areoptionally substituted with from one to four substituents selected fromthe group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino,mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; optionally, R² taken together with R³ form a fusedaromatic ring that is optionally substituted with from one to foursubstituents selected from halogen cyano, carboxy, sulfo, hydroxy,amino, mono- or di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and(C₁-C₆)alkoxy; the subscript p is an integer of from 1 to 3; W is a di-,tri- or tetra-valent linker which is acyclic, cyclic, aromatic or acombination thereof, having from 4 to 50 atoms selected from the groupconsisting of C, N, O, P and S and exculsive of hydrogen atoms that fillavailable valences, and further having a nitrogen atom directlyconnected to the adjacent carbonyl group; K is selected from the groupconsisting of a lone pair of electrons, H, OH, SH, NH, (C₁-C₈)alkyl,aryl, an amino protecting group and and a hydroxy protecting group; thesubscript n is 0 or 1; and when n is 1, Y is a cleavable linking groupand L is a solid support; and when n is 0, L is a phosphoramidite orreactive functional group the subscript p is an integer of from 1 to 3.53. An oligonucleotide probe having an attached 5,6-dichlorofluoresceindye selected from the formulae:

wherein, R^(x) and R^(y) are H or independently selected protectinggroups; R^(1′), R¹, R², R³and R⁴ are each independently selected fromthe group consisting of H, halogen, cyano, CF₃, (C₁-C₈)alkyl,(C₁-C₈)alkylthio, (C₁-C₈)alkoxy, aryl and heteroaryl; wherein the alkylportions of any of R^(1′) and R¹ through R⁴ are optionally substitutedwith halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy,cyano, haloacetyl or hydroxy, and the alkyl portions of the substituentshave from 1 to 6 carbon atoms; and the aryl portions of any of R^(1′)and R¹ through R⁴ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; optionally, R² takentogether with R³ form a fused aromatic or heteroaromatic ring that isoptionally substituted with from one to four substituents selected fromhalogen cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;and tautomeric forms thereof; and the wavy line indicates the point ofattachment to said oligonucleotide or a linking group joining saidoligonucleotide to said dye.
 54. An oligonucleotide probe in accordancewith claim 53, wherein said 5,6-dichlorofluorescein is selected from thegroup consisting of:


55. An oligonucleotide probe in accordance with claim 53, furthercomprising an attached quencher.
 56. An oligonucleotide probe inaccordance with claim 53, further comprising an attached minor groovebinder.
 57. An oligonucleotide probe in accordance with claim 53,further comprising an attached quencher and a minor groove binder,wherein said dye is attached at the 3′-end of said oligonucleotide probeand said quencher and said minor groove binder are attached at the5′-end of said oligonculeotide probe.
 58. An oligonucleotide probe inaccordance with claim 53, further comprising an attached quencher and aminor groove binder, wherein said dye is attached at the 5′-end of saidoligonucleotide probe and said quencher and said minor groove binder areattached at the 3′-end of said oligonculeotide probe.